AU2004201216A1 - Hybrid expression of Neisserial proteins - Google Patents
Hybrid expression of Neisserial proteins Download PDFInfo
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- AU2004201216A1 AU2004201216A1 AU2004201216A AU2004201216A AU2004201216A1 AU 2004201216 A1 AU2004201216 A1 AU 2004201216A1 AU 2004201216 A AU2004201216 A AU 2004201216A AU 2004201216 A AU2004201216 A AU 2004201216A AU 2004201216 A1 AU2004201216 A1 AU 2004201216A1
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Landscapes
- Peptides Or Proteins (AREA)
Description
*I
1 HYBRID EXPRESSION OF NEISSERIAL PROTEINS The present application is a divisional of Australian Patent Application No.
2001239478, the entire contents of which are incorporated herein by reference. All documents cited herein are incorporated by reference in their entirety.
TECHNICAL FIELD This invention is in the field of protein expression. In particular, it relates to the heterologous expression of proteins from Neisseria N.gonorrhoeae or, preferably, N.meningiddis).
BACKGROUND ART International patent applications W099/24578, W099/36544, W099/57280 and WOOO/22430 disclose proteins from Neisseria meningitidis and Neisseria gonorrhoeae.
These proteins are typically described as being expressed in E.coli heterologous expression) as either N-terminal GST-fusions or C-terminal His-tag fusions, although other expression systems, including expression in native Neisseria, are also disclosed.
It is an object of the present invention to provide alternative and improved approaches for the heterologous expression of these proteins. These approaches will typically affect the level of expression, the ease of purification, the cellular localisation of expression, and/or the immunological properties of the expressed protein.
DISCLOSURE OF THE INVENTION In accordance with the invention, two or more 3, 4, 5, 6 or more) proteins of the invention are expressed as a single hybrid protein. It is preferred that no non- Neisserial fusion partner GST or poly-His) is used.
This offers two advantages. Firstly, a protein that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem. Secondly, commercial manufacture is simplified only one expression and purification need be employed in order to produce two separately-useful proteins.
Thus the invention provides a method for the simultaneous heterologous expression of two 25 or more proteins of the invention, in which said two or more proteins of the invention are fused they are translated as a single polypeptide chain).
la The method will typically involve the steps of: obtaining a first nucleic acid encoding a first protein of the invention; obtaining a second nucleic acid encoding a second protein of the WO 01/64920 PCT/IB01/00420 -2invention; ligating the first and second nucleic acids. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.
Where just two proteins are joined, the hybrid protein can be represented simply by the formula NH 2 -A-B-COOH. A and B can each be selected from any Neisserial proteins, and in particular those represented by SEQ#s 1-4326. The method is well suited to the expression of proteins orfl, orf4, orf25, orf40, Orf46/46.1, orf83, 233, 287, 292L, 564, 687, 741, 907, 919, 953, 961 and 983.
The 42 hybrids indicated by in the following table of form NH 2 -A-B-COOH are preferred: A. ORF46.1 287, 741. 919 '953 961 983 0RF461 X X X X X X :287 X X X X X X 741 X X X X X X 919 X X X X X X 953 X X X X X X 961 X X X X X
X
983 X X X X X X Preferred proteins to be expressed as hybrids are thus ORF46.1, 287, 741, 919, 953, 961 and 983. These may be used in their essentially full-length form, or poly-glycine deletions (AG) forms may be used AG-287, AGTbp2, AG741, AG983 etc.), or truncated forms may be used AI-287, A2-287 etc.), or domain-deleted versions may be used 287B, 287C, 287BC, ORF461- 433 ORF46 43 3 6 08 ORF46, 961c etc.) and so on.
Particularly preferred are: a hybrid protein comprising 919 and 287; a hybrid protein comprising 953 and 287; a hybrid protein comprising 287 and ORF46.1; a hybrid protein comprising ORF1 and ORF46.1; a hybrid protein comprising 919 and ORF46.1; a hybrid protein comprising ORF46.1 and 919; a hybrid protein comprising ORF46.1, 287 and 919; a hybrid protein comprising. 919 and 519; and a hybrid protein comprising ORF97 and 225.
Further embodiments are shown in the drawings and include AG287-919, AG287-953, AG287-961, AG983-ORF46.1, AG983-741, AG983-961, AG983-961C, AG741-961, AG741-961C, AG741-983, AG741-ORF46.1, ORF46.1-741, ORF46.1-961, ORF46.1-961C, WO 01/64920 PCT/IB01/00420 -3- 961-ORF46.1, 961-741, 961-983, 961C-ORF46.1, 961C-741, 961C-983, 961CL-ORF46.1, 961CL-741, and 961CL-983.
Where 287 is used, it is preferably at the C-terminal end of a hybrid; if it is to be used at the N-terminus, if is preferred to use a AG form of 287 is used as the N-terminus of a hybrid with ORF46.1, 919, 953 or 961).
Where 287 is used, this is preferably from strain 2996 or from strain 394/98.
Where 961 is used, this is preferably at the N-terminus. Domain forms of 961 may be used.
Alignments of polymorphic forms of ORF46, 287, 919 and 953 are disclosed in WO00/66741. Any of these polymorphs can be used according to the present invention.
Preferably, the constituent proteins (A and B) in a hybrid protein according to the invention will be from the same strain.
The fused proteins in the hybrid may be joined directly, or may be joined via a linker peptide e.g. via a poly-glycine linker where n 3, 4, 5, 6, 7, 8, 9, 10 or more) or via a short peptide sequence which facilitates cloning. It is evidently preferred not to join a AG protein to the C-terminus of a poly-glycine linker.
The fused proteins may lack native leader peptides or may include the leader peptide sequence of the N-terminal fusion partner.
Host It is preferred to utilise a heterologous host. The heterologous host may be prokaryotic or eukaryotic. It is preferably E.coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonenna typhimuriumn, Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea, Mycobateria M.tuberculosis), yeast etc.
Vectors, hosts etc.
As well as the methods described above, the invention provides nucleic acid and vectors useful in these methods host cells containing said vectors proteins expressed or expressable by the methods compositions comprising these proteins, which may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions these compositions for use as medicaments as vaccines) or as diagnostic reagents (f) WO 01/64920 PCT/IB01/00420 -4the use of these compositions in the manufacture of a medicament for treating or preventing infection due to Neisserial bacteria a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria, and/or a reagent which can raise antibodies against Neisserial bacteria and a method of treating a patient, comprising administering to the patient a therapeutically effective amount of these compositions.
Sequences The invention also provides a protein or a nucleic acid having any of the sequences set out in the following examples. It also provides proteins and nucleic acid having sequence identity to these. As described above, the degree of 'sequence identity' is preferably greater than (eg. 60%, 70%, 80%, 90%, 95%, 99% or more).
Nomenclature herein The 2166 protein sequences disclosed in W099/24578, W099/36544 and W099/57280 are referred to herein by the following SEQ# numbers: Application Protein sequences SEQ# herein W099/24578 Even SEQ IDs 2-892 SEQ#s 1-446 W099/36544 Even SEQ IDs 2-90 SEQ#s 447-491 Even SEQ IDs 2-3020 SEQ#s 492-2001 W099/57280 Even SEQ IDs 3040-3114 SEQ#s 2002-2039 SEQ IDs 3115-3241 SEQ#s 2040-2166 In addition to this SEQ# numbering, the naming conventions used in W099/24578, W099/36544 and W099/57280 are also used 'ORF4', 'ORF40', 'ORF40-1' etc. as used in W099/24578 and W099/36544; 'm919', 'g919' and 'a919' etc. as used in W099/57280).
The 2160 proteins NMBo000 to NMB2160 from Tettelin et al. [Science (2000) 287:1809- 1815] are referred to herein as SEQ#s 2167-4326 [see also WOOO/66791].
The term 'protein of the invention' as used herein refers to a protein comprising: one of sequences SEQ#s 1-4326; or a sequence having sequence identity to one of SEQ#s 1-4326; or a fragment of one of SEQ#s 1-4326.
The degree of 'sequence identity' referred to in is preferably greater than 60%, 70%, 80%, 90%. 95%, 99% or more). This includes mutants and allelic variants see WO 00/66741]. Identity is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affme gap search with parameters gap open penalty=12 and gap extension penalty=l. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.
The 'fragment' referred to in should comprise at least n consecutive amino acids from one of SEQ#s 1-4326 and, depending on the particular sequence, n is 7 or more 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more).
Preferably the fragment comprises an epitope from one of SEQ#s 1-426. Preferred fragments are those disclosed in WO 00/71574 and WO 01/04316.
Preferred proteins of the invention are found in N. meningitidis serogroup B.
Preferred proteins for use according to the invention are those of serogroup B.
N. meningitidis strain 2996 or strain 394/98 (a New Zealand strain). Unless otherwise stated, proteins mentioned herein are from N.meningitidis strain 2996. It will be appreciated, however, that the invention is not in general limited by strain. References to a particular protein '287', '919' etc.) may be taken to include that protein from any strain.
It will be appreciated that references to "nucleic acid" includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
BRIEF DESCRIPTION OF DRAWINGS Figures 1 to 26 show hybrid proteins according to the invention.
MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1 HYBRIDS OF ORF46 The complete ORF46 protein from N meningitides (serogroup B, strain 2996) has the following sequence:
LGISRKISLILSILAVCLPMHAJ{ASDLANDSFIRQVLDRQHFEPDGKY{LF
GSRGELAERSGHIGLGKIQSHQLGNLMIQQAAIKGNIGYIVPYSDHGHEV
HSPFDNHASHSDSDEAGSPVDGFSLYRIHWDGYEHHPADGYDGPQ.GGG
Y
WO 01/64920 WO 0164920PCT/IBO1/00420 PAPKGARDfIY SYDIKGVAQN IRLNLTDNRS TGQ1RLADfRFH GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE ISEGSNIAVM.HGLGLLSTEN KMARINDLAD MAOLKDYAAA NAAQGIEAVS NIFXAAIPIK GIGAVRGI(YG LGGITAHPIK KGKSAVSDNF' AflAAYAI(YPS PYHSRNIRSN LEQRYGKENI KNVKLADQRH PKTGVPFDGK GFPNPSKHVK YDTKLDIQEta VSDAKPRWIEV DRKTNK IMTTR EQVEXNVQEI RNGNRNSNFS KLKSAflBINF ADGMGKVTDS MNDKAFSRLV KSVKENGFTN KAYIVRGNNR VFAAEYLGRI BHELKKVDF PVPNTSWKNP
KRPRYRSK*
NAGSMLTQGV
IVGAGDAVQG
AIRDWAVQNP
RSQMGAIALP
TSSTVPPSNG
SGGGIPKAKP
QHXQLEIREIN
PWVEYVEING
TDVLNESGNV
The leader peptide is underlined.
The sequences of ORF46 fr-om other strains can be found in W000/66741.
ORF46 has been fused at its C-terminus and N-terminus with 287, 919, and ORFi. The hybrid proteins were generally insoluble,, but gave some good ELISA and bactericidal results (against the homologous 2996 strain): Protein ELISA Bactericidal Ab Orf 1-0rf46. 1-H-is 850 256 919-0rf46.1-His 12900 512 919-287-0rf46-His n.d. n.d.
0rf46.1-287His 150 8192 0rf46. 1-9 l9His 2800 2048 0rf46.1-287-9199His 3200 16384 For comparison, 'triple' hybrids of 0RF46.l, 287 (either as a GST fusion, or in AG287 form) and 919 were constructed and tested against various strains (including the homologous 2996 strain) versus a simple mixture of the three antigens. FCA was used as adjuvant: BZ232 MC58 NGH38 F6124 BZ133 Mixture 8192 256 512 1024 >2048 >2048 0RF46.1-27-9l9his 16384 256 4096 8192 8 192 8192 AG287-919-0RF46.lhis ,8192 164 .4096 18192 8192 116384 AG287-0RF46.1-9l9his 14096 1128 1256 18192 1512 11024 Again, the hybrids show equivalent or superior immunological activity.
Hybrids of two proteins (strain 2996) were compared to the individual proteins against various heterologous strains: 1000 MC58 F6124 (MenA) 0RF46.1-H1is <4 4096 <4 ORMi-Eis 8 256 128 ORFI-RF46.1-His ,1024 1512 1024 Again, the hybrid shows equivalent or superior immunological activity.
WO 01/64920 WO 0164920PCTIB01OO420 -7- Example 2 hybrids ofAG287 The deletion of the (GlY)6 sequence in 287 was found to have a dramatic effect on protein expression. The protein lacking the N-terminal amino acids up to GGGGGG is called 'AG287'. In strain M058, its basic sequence (leader peptide underlined) is: SPDVKS AflTLSKPAAP VVSERETEA1 EDAPQAGSQG QGAPSAQGSQ DNAAVSEENT GNGGAVTADN PKNEDEVAQN DMPQNAAGTD SSTPNHTPD)P NMLAGNMENQ ATDAGESSQP *ANQPDMAI4AA DGMQGDDPSA GGQNAGNTAA QGAQAGNNQ AAGSSDPIPA SNPAPANGGS NPGRVDLANG VLIDGPSQNI TLTHCKGDSC SGNNFLDEEV QLKSEFEKLS DAflKISNYK DGKNDKFVGL VAflSVQb2KGI NQYXIrFYXPK PTSFARPRRS ARSRRSLPAE M~PLIPVNQAfl TLIVDGEAVS LTGHSGNIFA PEGNYRYLTY GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY *NGEVIMFHTE NGRPYPTRGR FAAKVDFGSK SVDGIIDSGD DLH1MGTQKFK AAIDGNGPRG TWTENGSGDV SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEQD* AG287, with or without His-tag ('AG287-His' and 'AG287K', respectively), are expressed at very good levels in comparison with the '287-His' or '287 nae, On the basis of gene variability data, variants of AG287-His were expressed in Excoli from a number of MenB strains, in particular from strains 2996, MC58, 1000, and BZ232. The results were also good each of these gave high ELISA titres, and also serum bactericidal titres of >8192. AG287K, expressed from pET-24b, gave excellent titres in ELISA and the serum bactericidal assay.
Deletion of poly-Gly sequences is also applicable. to Thp2 (NMO46O), 741 (NMB 1870) and 983 (NMB 1969). When cloned in pET vector and expressed in Excoli without the sequence coding for their leader peptides and without poly-Gly as "AG forms"), the same effect was seen expression was good in the clones carrying the deletion of the poly-glycine stretch, and poor or absent if the glycines were present in the expressed protein.
AG287 was fused directly in-frame upstream of 919, 953, 961 (sequences shown below). and 0RF46.1: AG287-919
ATGTGCCAGTATGCGCCCGCAACGCGTCGTTGTAAAAAAA
GTAAAGTCCAAGAGTTAGAAGGGGCTCCCAGACAGTTGGC
GTTGCGAAAAGATGGTCGACAGAAACAAAGAAGrGACCAA
GAAGCCAATCCGACGAACACGGACACACGCATTCGTCGCC
GCTAACTCCTCATGGTGATTGAGGTATGTAWGTTATACG
CCTGAATTAGTACATTAGCATCTTAGTAATTTGAGAAGAC
TCAACGATGAATAAGGCGAGATGGATTAAAAGGAACAAAT
ACATTGTCAACGTAGTAGACACAAAGCTATAAAAAGCGTC
TCTACGGGTCGCTCGAGTGGAGTGTCTCGGTCGTACCGCA
CAGGAAGTATTGTGGACGCGCTAGGCTCGCAACTGGCGAG
AATCGTTTATAGGCGAATGCGGGACTTcccGGGAGCACGC
AAGCAAGTGTGAGCGGAACGGAGGTCTTCTCGAAGCGCGA
CCATGGCGTTCGAAGCATCGAGAACGGAGCTACAACGGTA
TGCATATGGGTACGCAAAAATTCAGCCGCCATCGATGGCGGCTTAGGGCTTGGGGAAAAGGCGGC
GGGTTTCGAGTTCGCGCGCAGAGGCGAATcGTTGCGCGTC WO 01/64920 PCTIIB01/00420 -8-
GAAGGGATGCTTTCGCAAAACAGTGTCGGAGGAGCAGAGG
ATCACTTCCACGCCTCTACAGCCGCGCGTGCTCCAC~.CG
ACAGTGCGGCGGCTTTCGTTCGACGCCGCCCGGGCCGATT
GCAAGCGATCTCCTGCGGCATGAACGCAGTGAGTTwGGCA
GCTTAACCGCATCTCGCAAATTTTACCATCCCGGCGTGAG
AAGAGCTCGTCGTCGCATCACCGGTAGGGCAAGGAGCCAC
CGTCCATAGTTCCAGTTACCGTCCTCTCGTTCGGGAAGCT
GTCCTAGAAGGAAAACGAATGCAACGGCCCTCGCACCCCA
I GGC
CATACGGCCCTAGCAGCCAATGTAGCAGCCGCACTTTTT
CAACAGCCGCGCGA
CCGCGCAAAACGACGTTCGCAA~,AA
CCTCTTCTGAGTTTGGAALGCACCACCGCGcTGTCGGACA
GCTTTCGAATCCAGCCCGAT'TGTAACCACAACTTCGGGT
TAMCTCGAG
1 1{ASPDVI(SAD TLSKPAAPVV AHKETEVKE .D.APQAGSQGQG APSTQGSQDM 51 AAVSAENTGNGGAATTDKPK NEDEGPQNDM PQNSAESMNQ. TGINNQPADSS 101 DSAPASNPAP ANGGSNDFGRV DLANGVLEIDG PSQNITLTHC KGDSCNGDNL 151 LDEEAPSKSB PENLNESERI EI(YIGDGKSD KPTNLVATAV QANGTNKXVI 201 IYRDKSASSS SARPRRSARS RRSLPAEKPL IPVNQAnTLI VDGEAVSLTG 251 HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEN I.AGTAVYNGE 301 VLHPHTENGR PYPTRGRPAA KVDFGSKSVD GXZOSGDDLH MGTQXFKAAI 351 DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVPAG.
401 KKEQDGSGGG GCQSKSIQTF PQPDTSVING PDRPVGIPDP AGTTVGGGGA 451 VYTVVPHLSL PUWAAQDFAX SLQSFm.,GCA NLIUNRQGWQD VCAQAFQTPV 501 HSFQAI(QFF RYFTPWQVAG NGSLAGTVTG .VYEPVLKGDD RRTAQARP 551 YGIPD)DFISV PLPAGLRSGK ALVRIRQTGI( NSGTIDNTGG THTAflLSRFP 601 ITARTTAIKG P.FEGSRFLPY HTRNQINGGA LDGKAPILGY AEDPVELFFM 651 HIQGSGRLKT PSGKYIRIGY ADKNEHPYVS IGRYMADKGY LXLGMTSMQG 701 IKAYMQNPQ RLaEVLGQNP SYIFFRELAG SSNDGPVGAL GTPLMGEYAG 751 AVDRHYITLG APLFVATAHP VTRKAI.NRLI MAQDTGSAIK GAVRVDYFWG 801 YGDEAGELAG KQKTTGYVWQ LLPNGMKPEY RP* AG287-953
ATGTGCCAGTATGCGCCCGCAACGCCCTTGTCGAAAAAAA
GTAAAGTCCAACCGTCCAGCGGGGCTCCCAGACAGTTGGC
GTTGCGAAAAGATGGTCGACACGCACCAATAGCAGACCAA
GAAGCCAATCCGACGAACACGCACACACGCATTCGTCGCC
GCTAACTCCTCATGGTC=TTGAGTGTTGTAGCTTGTGTG
CCTGAATTAGTACATTACCATCTTAGTAALTATGTAGACCG
TCAACGATGAATAAGGCGAGATGGATTAAAAGGAACAAAT
ACATTGTCAACGTAGTAGACAAAAAGCTATAAAAAGCGTC
TCTACGGGTCGCTC(CCGCAGGTGTCTCGGTCGTACCGCA
CAGrGT~,TATTGTGGAC~TACCGCGGATCGATTTCCCCA.G
AATCGTTTATAGGCGAATGCGGGACTTCCCGGGAGCACGC
AAGCAAGTGTGAGCGGAACGGAGGTCTTCTCGAAGCGCGA
CCATGGCGTTCGAAGCATCGACATTTGCGATTGCGGCArA TTrAAGGAGAAATAACGCTGT~.ACGTTAGGCTGCGAAGCG
GGGTTTCGAGTTCGCGCGCAGAGGCGAATCGTTGCGCGTC
GAAGGGATGCTTTCGCAAAACAGTGTCGGAGGACACAAAT
GAGAACCCACCCTTGCTGCATTACCACCACTGCGTTAGTT
ACGTCGCATc.cAGAAAGGCGAATGC~-CACCGT~ACTcA~
GGTGACCTACACCTAACGCAATTCAGCCCAACGAACGTTT
TCACATCATCAGCAALCGTTCTGCGACTACTCCGAACGCC
GTAATA-GCAAATACGTCALGCCAGCAAcGA~Tc~GC~TcG
ACACTGCGACATGGGGATCTGTACTGTTACAACTCCTGCT
CAAATCGAGGCAGOACATAACTGAG
1 l4ASPDVKSAfl TLSRPAAPWV AEKETEVKED APQAGSQGQG APSTQGSQDM.
51 AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPAflSS 101 DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL 151. LDEEAPSRSE FENLNESRI EKYKKDGKSD KP'INLVATAV QANGITqKYVI WO 01/64920 PCT/IBO1/00420 201 IYRDKSASSS SARPRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG 251 HSGNIFAPEG NYRYLTYGAE IC.PGGSYALR VQGEPAXGEM LAGTAVYNGE 301 VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKrKAAI 351 DGNGFI(GTWT ENGGGDVSGR FYGPAGEEVA GXYSYRPTDA EKGGFGVPAG 401 RREQDGSGGG GATYKVU)EYH ANARPAIDHF NTSTNVGGFY GLTGSVEFflQ 451 AXRDGKIDIT IPVANLQSGS QHFTDHLKSA DIFDAAQYPD IRFVSTRFNF 501 NGKKLVSVDG NLTMHGKTAP VKLKAEKFNC YQSPMAKTEV CGGDFSTTID 551 RTKWGVDYLV NVGM4TKSVRI DIQIRAAXQ* AG287-961
ATCACCGTTAACG~.CCCGCAACGCGTCGTTGTAAAAAAA
GTAAAGTCCAAGAGTTAGAAGGGGCTCCCAGACAGTTGGC
GTTGCGAAAAGATGGTCGACA
GAAACAAAGAAGGGCGAAA
GAAGCCAATCCGACGAACACGGACACACGCATTCGTCGCC
GCTAACTCCTCATGGTGATTGGAGTGTTGTAGCTTGTGTG
CCTGAATTAGTACATTA~.GTCTTAGTAATTTGAGAAGAC
TCAATAATTAATTATATTACAATAATTAAAAGGAACAAAT
ACATTGTCAACGTAGTAGACAAAAAGCTATAAAAAGCGTC
TCTACGGGTCGCTCGAGTGGAGTGTCTCGGTCGTACCGCA
CAGGAAGTATT'AGGAGGTACCGCGGATCGAACTGGCGAG
AATCGTTTATAGGCGAATGCGGGACTTCCCGGGAGCACGC
AAGCAATCTCGCCGCTTCAGCAGGTCTTCTCGAAGCGCGA
CCATGGCGTTCGAAGCATCGAGAACGGAGCTACAACG~
A
TTGTCCAAATAACGCACAGAAGGTTAGGCTGAGAATGW
GGGTTTCGAGTTCGCGCGCAGAGGCGAATCGTTGCGCGTC
GAAGGGATGCTTTCGCAAAACAGTGTCGGAGGACAAAGCA
GAGTAAACGCCG~.CTGTCGCAACAGCAGATACGTCAGTG
r.GCACAGCTGTAGCGAATACAAAAGATCGCAGTAGCAGC
TTAAGCGGCGAAATGGCACTACXLACTATAACACAAGCA.TC
AAGAAGTCGACGATGAATACACAGTGAAATAGCCTACGTC
GAGCCCGAGACACAGCTATATTGAAATTAGCTTCGAAATA
ACATTGAAATAGAATAAGCTGTAACTGCACTCG~.ATACA
ATG~.TCTrGTAACAATAGAAGACGCAA~,CAGACAAAAGC
GAGACAAAACTGTCAATAACGCGACGACGCAGCAGTCGTG
ACGTAATCGCAAGCGACGCCGCAATACAACAGTAACCAGA
AAGTAATCAAAGAAATCGCTTAACGGAATTAACATTTAAT
GAGTTACCATCGAATGAAAGTTGTCGTAAACATCGTAGTC
CGCGAGTTGTAAATTAACGGAAGACCCAGCTGAACACGGT
TCGTTTCACTAACTGTOTCAGTAGCGATGCPTCATCATGC
GTGCTGTCGCTCCTACAACTGCCALGAGGGCGCGATCTCG
TCTTCCGCAGCCTACCATGTCGGCGTCATTACGAGGTIACTCGAG'
I HASPDVI(SAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM 51 AAVSAENTGN GGAA'ITDKPK NEDEGPQNDI4 PQNSABSANQ TGNNQPADSS 101 DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGI2SCNGDNL 151 LDEEAAPSKSE FENLNESERI EKYKKflGKSD KFTNLVATAV QANGTNKYVI 201 IYKDKSASSS SARPRRSARS RRSLPABMPL IPVNQAflTLI VDGEAVSLTG 251 HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAI(GEM LAGTAVYNGE 301 VLHFHTENGR PYPTRGRFAA KVflFGSKSVD GIIDSGDDLH MGTQKFKAAI 351 DGNGPKGTWT ENGGGDVSGR F'YGPAGEEVA GKYSYRPTDA EKGGFGVFAG 401 KREQDGSGGG GATNDDDVXK AATVAIAAAY NNGQEINGPK AGETIYDIDE 451 DGTITKKDAT AAflVEADDFK GLGLRKVVTN LTKTVNENKQ NVDAKVKAAE 501 SEIEKLTTRL ADTDAALAflT DAALDATTNA LNKLGENITT RAEETKTNIV 551 KIDEKLEAVA D VDKHAEA NDIADSLDET NTKADEAVKT ANEAKQTAE 601 TKQNVDAKVK AAETAAGXAE AAAGTANTAA DKARAVAARV TDIKAflIATN 651 KDNIADCANS ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH 701 DTRLNGLDKT VSDI*RKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK 751 SESAVAIGTG FRFTMEFAAK AG;VAVGTSSG SSAAYHVGVN YEW* WO 01/64920 PCTIBOI/00420 ELISA Bactericidal AG287-953-H1is 3834 65536 AG287-961-His 108627, 65536 Tlie bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens (using 287-GST) for 919 and 0RF46. 1: SMixture with 287 Hybrid with AG287 9191 32000 128000 0RF46.lj 128 16000 Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained: 919 0RF46.1 Strain Mixture Hybrid Mixture Hybrid NGH38 1024 32000 16384 MC58 512 8192 512 BZ232 512 512- MenA (F6124) 512 32000 8192 MenC (C11) >2048 1>2048
I-
MenC (BZ133) >4096 164000_ 18192 The hybrid proteins with AG287 at the N-terminus are therefore immunologically superior to simple mixtures, with AG287-0RF46. 1 being particularly effective, even against heterologous strains. AG287-0RF46. 1 K may be expressed in pET-24b.
The same hybrid proteins were made using New Zealand strain 394/98 rather than 2996: AG287NZ-919
ATGGCTAGCCCCGATGTCAGTCGGCGGACACGCTGTCACCTGCCGCCCCTGTTGTCTAAGAGACAG
GCAGAGTCCAAGAGTTAGAAGGGGCTCCCAGGThGTTGGC
GTTGAGAAAAGATGGTCGACACGCACCAATAGCAGGCCAA
GAAGCCA6TCGCAAAAATTAACATAACCGTCATTCCGCGM
ATAACAGACGTCGGATGACGCGCACACCAAGCAAAGCGCG
ATCGGGCACGCGAGGGAATCGG~TCGTCCAGAAACACGAA
AACACGCGTTAATCGCCTACAACTGGCCATGGTGGTTGAG
ACAGGGATCGTTATACGCGCGAATTAGTACATTAGCATTG
AGGCA~TTTGAGAAGAACAATCGATGAATAGGTCGCAAAG
AATCAkAAWAGAGCGAGAGTATTTGTTGTCGTGGGAAGA r.ATATATTTACTTTACTAACATCTTcCATAGrTCGAGTGG
CGTGTCGCGGTCGTATCGCACAGGAAGTATTGTGGACGCG
CTAGGCTCGCAACTGGCGAGAATCGTTTATAGGCGAATGC
GGGACTTCCCGGTAGCACTCAAGCAAGTGGGAGCGGAACG
GAGGTCTTCTCGAAGCGCGCCCTCGGCGTTCGAAGCATCG
AGCAAATCTGTGGACGGCATTATCGACAGGGCGATGGTTTGCATATGGGTACG
TTCAAGCCGCCATCGAT
GGACGTTAGGCTGCGAAGCGGGGTTTCGAGTTCGCGCGCh WO 01/64920 PCT/IBOI/00420
GGCCCGGACCGGCCGGTCGGATCCCCGACCCCGCCGACGACGGTCGGCCGGCGGGCCGTCTATACCGTTGTA
CCCCTTCTCCATGC*CCGATCCAAGCGATCTCCTG
CTCCCA
TTAAACCAGCGCGAGGGGCAGCTTAACCGCATCTCGCAA
TTTTACCATCCCGGCGTGAGAAGAGCTCGTCGTCGCATCA
CCGGTArGCAGCGC.AGCCACCGTCCATAGrTTCCAGTTACC
GTCCTCTCGTTGGACGAACCTGCGACGCGCGGAAAACGAAT
GAATCGCGAAAACCGCTTCGTTCCTACCCCCAGCACAGCG
TTGAGACGTCTCCACCCCCACATACGGCCCTAGCAACCGT
CTGTAGCAGCCGCATTTTAGAACAGCCGCGCGAACCTCG
AATCTCCTGCAGCAAAAGAACCTCTTCTGAGTTTGGAAAG
TACCACCGCGCTGTCGGACAGCAAGGCAATCCAGCCCGAT
TTGTAACCACAACTTCGGGTGCGAGACAGCGCCTGCCCGG
ACCGTAGGGAAGCGGATGCGCATCTACTGCCCCTTTTGCC
GCCTCGTCCCAGCTACGCGTATGGAGTCGCGGGTAAGGGT
CGGGATTTTGGTCGGCACGCACTCGCACGAACCGTAGCG
CAGCTCCTACCCAACGGTATGAAGCCCAATACCGCCCGTAAAAGCTT
1 I4ASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGGAPSAQGGQDM 51 AAVSEENTGN GGAAAT'DKPK NEDEGAQNDM PQNAAflTDSL TPNHTPASNN 101 PAGNMENQAP DAGESEQPN QPDMANTAflG MQGDDPSAGG ENAGNTAAQG 151 TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL 201 THCKGDSCSG NNFLDEEVQIJ KSEFEXIJSDA DKISNYKKDG KNDGKNDKFV 251 GLVAflSVQMK GINQYIIP'YK PKPTSPARFR RSARSflRSLP AEMPLIPVNQ 301 AD'rLIVDGEA VSLTGHSGNI FAPEGNJYRYL TYGAMCLPGG SYALRVQGEP 351 SKGEMLAGTA VYNGEVLHFH 'rENGRPSPSR GRPAAKVDFG SIKSVDGIIDS 401 GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY 451 BPTDAEKGGF GVFAGKKEQD GSGGGGCQSI( SIQTFPQPDT SVINGPDRPV 501 GIPDPAGTTv GGGGAVYTVV PHLSLPHWAA QDFAICSLQSF RLGCANLKNR 551 QGWQDVCAQA FQTPVHSFQA KQFFERYFTP WQVAGNGSLA GTVTGYYEPV 601 LKGDDRPRTAQ ARPPIYGIPD DFISVPLPAG LRSGKALVRI RQTGKINSGTI 651 DNTGGTHTAD LSRFPITART TAIKGRFEGS RPLPYHTRNQ INGGALDGKA 701 PILGYAEDPV ELFFMHfIQGS GRLKTPSGKY IRXGYAflKNE HPYVSIGRYM 751 ADKGYLKLGQ TSMQGIKAYM RQNPQRLAEV LGQNPSYIFF RELAGSSNDG 801 PVGALGTPLM GEYAGAVDRH YITLGAPLFV ATAH-PVTRKA LNRLIMAQDT 851 GSAIKGAVRV DYFWGYGDEA GELAGKQKTT GYVWQLLPNG MKP3YRPp* AG287NZ-953 ATG ACCGTTAGCGGAAGTTAACTCGCCGTTTTAAAAAAA
GCAGAGTCCAAGAGTTAGAAGGGGCTCCCAGGTAGTTGGC
GTTGAGAAAAGATGGTCGACACGCACCA&TAGCAGGCCAA
GAAGCCAAGCCGTCGTGTGCCCATAACCGTCATTCGCGAA
ATO"CAGACGTCGGATGACGCGAACACGTTGAAACCGCC
ATCGGGCACGCGAGGGAATCGGATCGTCCAGAAACACGAA
AATCAAACCGCCGGTTCTCAAATCCTGCCTCTTCA ATCCTAGCGCcAACGGTGGTGATTTTGGAJG
ACGAACGTGGGCAATTCTGTGTGATTGACGGCCGTCGCATATAACGTTGACCCACTTA;AGGCGATTTGT
AGGCAATTTGAGAAGAACAATAATTAAATATAGAAAAAAG
AATCAAAAGGAATAGGAATAAATTTGTTGTCGTGGGAAGA
GGAATCAATCAATATATTATCTTTTATAACCTAACCCACTTcATTTGrCCATTTAGGCGTCTCACGGCGAGG
CGTGTCGCGGTCGTATCGCACAGGAAGTATTGTGGACGCG
CTAGGCTCGCAACTGGCGAGAATCGTTTATAGGCGAATGC
GGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAZATGCTCGCGGGCACGGCAGTGTAC
AACGGC
GAGGTCTTCTCGAACGCTCTCCTCGGCGGTGCCAATGTTrG
AGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAATTCAAGCCGCCATCGAT
GGAAACGGCTTTAAGGGGACTTGGACGGAATGGCGGCGGGrATGTTCCGGAAAGTTTTACGGCCCGGCCGGCGAG GAGG~.GAAAACACCCAAAGr.AAGGGATGCTTTCGCA"rA
CAGTGTCGGAGGACACAAATGAGAACCCACCCTTGCTGCA
TTCAACACCAGCACCAACGTCGGCGGTTTTTACGGTCTGACCGG
CGTCGGTTCGACCCAAGCGACGGT
AAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGCrGTTCGCAACACTTTACCGACCACCTGAAATCAGCCGAC
ATTCAGCCCAACOAACGTTTTCACATCATCAGCAAATGTC
GTGCGACTACTCCGAACGCCGTAATAACGAMTACGTCA.G
CCGATGGCGAACCGAAGTTTGCGGCGGCGACTTCACCCACCATCGACCGCCCAATGGGcGTGACTACCTC GTTAACGITrGGTATGACCAAAGCGTCCGCATCGACATCCAATCAGGCAGCCAAACAATAAAGCTT 1 M~ASPDVKSAfl ThSKPAAPVV SURETEAKED APQAGSQGQG APSAQGGQDX WO 01/64920 PCT/IBOI/00420 AAVSEENTGN GGAAATDKPK NEDEGAQNDM PAG1NQAP DAGESEQPAN QPDI4ANTADG TNQAENNQTA GSQNPASSTN PSATNSGGDF THCKGDSCSO NNFLDBEVQL KSEFEKLSDA GLVADSVQbMK GINQXIImY PKPTSFARFR ADTLIVDGEA VSLTGHSGNI FAPEGN'YRYL SRGEMLAGTA *VYNGEVIMFH TEINGRPSPSR GDGLHMGTQK FKAAIDGNGP KGTWTE1NGGG RPTDAEKGGF GVFAGKKMQD GSGGGGATYK VGGFYGLTGS VEFDQAX1RDG KIDITIPVAN AQYPDRFVS TKFNFNGMK VSVDGNLTMH AKTEVCGGDF STTIDRTKWG VflYLVNVGMT PQNAAflTDSL TPNHTPASNM XQGI2DPSAGG ENAGNTAAQG GRTNVGNSWV IDGPSQNITL DKXSNYKRDG KNflGKNDRP'V RSARSRRSLP AEMPLIPVNQ TYGAEKLPGG SYALRVQGEP GRPAA~VDP'G SYSVDr.IIDS DVSGKPYGPA GEEVAGKYSY VDEYHAN'AIRF AIflHPITSTN IJQSGSQHPTD IUA(SADIFDA GKTAPVKLKA EKFNCYQSPM KSVRIDIQIE AAQ* £iG287NZ-961 ATGTGCCAGCATGCGCCCGCAACGCCCTTGTCGAAGrAAA
GCAGAAGGCCGCGTCCAGCGGGGCTCCCAGGTAGTTGGC
GTTGACkATCGCAGCGGGCGAAOAAACAAITAGCAGGCCAA
GAAGCCAAGCCGTCGTGTGCACATAACCGTCAAGCGCGM
ATGAkCAGACGTCGGATGACGCGAACACGTTGAAAGCGCG
ATCGGGCACGCGAGGGAATCGGATCGTCCAGAAACACGAA
AACACGCGTTAATCGCCTACAACTGGCCATGGTGGTTGAG
ACACTGCATTTGGTGCGCGCCAATTAGTACATTAGCATTG
AGGCAATTTGAGAAGAACAATCGATGAATAGGTCGCAAAG
AATCAAGT~-AGAGCGAGA"X
TTTGTTGTCGTGGGAAGA
GGAATCAATCAATATATTATCTTTTATAAACCTAACCCACTTCATTGCGCGATTTAGGCGTCTGCACGTGAGG
CGTGTCGCGGTCGTATCGCACAGGAAGTATTGTGGACGCG
CTAGGCTCGCAACTGGCGAGATACGACGCTCGGGAAATGC
GGGACTTCCCGGTAGCACTCAAGCAAGTGGGAGCGGAACG
GATCGATTAAGAACGCTdTCCTCGGCGTTCGAAGCATCG
AGAACGGAGCTACAACGGTGTTCTTGTCCAATCAGCCACA
GGACGTTAGGCTGCGAAGCGGGGTTTCGAG~VCGCGCGCA
GAGGCGAATCGTTGCACGTCGAAGGGATGCTTTCGCAAAA
CAGTGTCGGAGGACAAAGCAGAGTAAACGCCGGCATCGTC
TAACAWCAATACGTCAGTGGAACTTCAATAGAAGCCATC
AAAAAGACGACGTTGACGCATTAGTTGTTAAAGCTATACG
ACAACGCAGAAAAAACTGTCAAGAAGTCGACGATGAATAC
ACAGTGAAATAGCCTACGTCGAGCCCGAGACACAGCTATA
TTGAAATTAGCTTCGAAATAGCATTGAAATAGAATAAGCT
GCGTCGCAAGAGCAGATACAACCGTCTCGTAACAATAGAA
GA(CGCAACCATAGCACGCGCAGACAAAACTGTCAATAAC
GCGACGACGCAGCAGTCGTGACGTAATCGCAAGCGACGCC
GCAATA~.CTAACGTTGTCACAGTAATCAAACAAATCGCT
TAACGGAATTAACATTTAATGTGCGAGTCACAAA~.AAAG
TTGTCGTAAATCTGCACCAATGCGAGTTGTAAATTAACGG
AAGACCCAGCTGAACACGGTTCGTTTCACTAACTGTGTCA
GTAGCGATGCGTCATCATGCGCCACGACGTCGTTCGALCT
GCGCAGAGGGCGCGATCTCGTCTCCGCACTTGCTATAGGG
TAAAAGCTT
M4ASPDVKSA.D TLSKPAAPVV SEKETEAKED APQAGSQGQG ASAQGGQDM AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM PAGNMENQAP DAGESEQPAN QPDMANTAflG MQGDDPSAGG ENAGNTAAQG TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL THCKGDSCSG NNFLDEEVQIJ KSEFEKLSDA DKISNYKKDG KNDGKNWKFV GLVADSVQI4K GINQYIIFYR PKPTSFARFR ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GDGLHMGTQI( FKAAIDGNGF KGT1WTENGGG RPTDAEKGGF GVFAGKKEQD GSGGGGATND INGFKAGETI YDIDEDGTIT KKI2ATAAflVE NENKQNVDAI( VKAAESEIEK LTTKLADTDA ENITTFAEET KTNXVKIDEK LEAVAflTVDK EAVKTANEAI( QTAHETKQNV DAKVKAAETA VAAKVTDIKA DIATNKDNIA KKANSAflVYT LDTRLASAEK SIhflHDTRLN GLDKTVSDLR NVGRFNVT]AA VGGYKSESAV AIGTGFRFTE
IWGVNYLPW*
RSARSRRSLP
TYGAERLPGG
GRFAAXVDFG
DYVSGKFYG;PA
DDVXKAAVA
ADDFKGLGLK
ALADTDAALD
HAEAPNDIAD
AEMPLIPVNQ
SYALRVOGEP
SKSVDGIIDS
GEEVAG1KYSY
IAAAYNNGQE
RVVTNLTKTV
ATTNALNKLG
SLDETNTFAD
AX3KAEAAAGT ANTAADKABA 1RBESDSKFVR IDGLN'ATTEK KETRQGLAEQ AALSGLFQPY NFAAKAGVAV GTSSGSSAAY WO 01/64920 WO 0164920PCT/TBO1/00420 Example 3 -hybrids of4JG983 Protein 983 has the following sequence: 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 9.51 1001 1051 MTTPTFPTK TFKPTAMAI.A VATTLSACLG NSRATTASA AVSYAGI:KN MCKDRSMLCA LHTGDFPNPN DAYKNLINIJX PA MAGYTGR LYGRREHGYN ENYIGNYTAYM RKEAPEDGGG DIRHVKEIGH IDLVSHIIGG RSVDGRPAGG MVAAIRNAWV KLGERGVRIV ]NNSFGTTSRA DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK LLPFYEIWAQ KGIITVAGVD RSGEKEM MWCLSAPYEA SVRPTRTNPI QIAGTSFSAP LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK SDIAYSFUND ISGTGGtLIKK GGSQLQLHGN KSDMRVETKG ALXYNGAASG GSLNSDGIVY LDGKGTLYTR LGKIJLKVDGT AIIGGKLYMS AAKIGQDYSF PTNIETD= L LASLDSVEKT ASAAAHSAPA GLKHAVEQGG SNLENL!4VEL DMPGIRPYGA TFRAAAAVQH ANAADGVRIF RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW KTGENTTAAA TLGXGRSTWS ENSANAKTDS FSYGRYKNSI SRSTGADEHA EGSVNGTLsMQ GGLRYDLLKQ DAFAEKGSAL GWSGNSLTRG FATAGVERflL NGRDYTVTGG FTGATAATGK PO G983 GGGGGTSAPD FNAGGTGIGS GRDDVAVTDR DAKINAPPPN GVEVGIVDTG ESVGSISFPE KDIEASFDDE AVIETEAKPT IAPDATLHflM NTNDETKNqEM GTADLFQIAN SEEQYRQALL NMLFIFST.GN DAQAQPNTYA YGEPGTBPLE YGSNHCGITA IVTGTAALLL QKYPWMSNDN AMNGPASFPF GDFTADTKGT
NTYTGKTIIEGGSLVLYGNN
LADTDQSGN ETVHIKGSLQ
ARGKGAGYLN
AGSEGDTLSY
DASESSATPE
NSLAATVAD
EQGGVEGKtMR
ISLFAGIRHD
LGALGGVNVP
TLVGLAGLRL
TGA1UNMPHTR
STGRRVPFLS
YVRRGNAART
TVEI'AAAflRT STAAHAflMQG
GSTQTVGIAA
AGDIGYLKGL
FAATGDLTVE
SQPLSDKAVL
LVAGLGADVE
FGNGWNGLAR YSYAGSXQYG NHSGRVGVGY R?* AG983 thus has the following basic sequence: TSAPD FNAGGTGIGS GRDDVAVTDR DAKINAPPPN NSRATTACSA AVSYAGIKNE MCKDRSMLCA
LHTGDFPNPN
LYGRXEHGYN
DIRHVXEIGH
MVAAIRNAWV
DAYKNLINLX(
ENYKNYTAYM
IDLVSHIIGG
KLGERGVRIV
PAIEAGYTGR GVEVGIVDTG RKEAPEDGGG KDIEASFDDE RSVDGRPAGG IAPDATLHn.! NNS9GTTSRA GTAflLFQIAN jj DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK NMLFIFSTGN LIJPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE MWCLSAPYRA SVRFTRTNPI QIAGTSFSAP IVTGTAALLL LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK AMNGPASFPF SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGKTIIE KSIX4RVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGN LDGKGTLYTR LGKLLKVDGT Al IGGI(LYMS A.RGKGAGYhN AIGQDYSF FTNIETDGGL LASLDSVEKT AGSEGDTLSY ASAAARSAPA GLKHAVEQGG SNLENMXTEL DASESSATPE DMPGIRPYGA TFRAAAAVQH ANAAflGVRIF NSLAATVYAfl RRLKAVSDGL DHI'GTGLRVI AQTQQDGGTW EQGGVEGKMII KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIPJID FSYGRYIKNSI SRSTGADEHA EGSVNGTLMQ LGALGGVNVP GGLRYDLLKQ DAPAEKGSAL GWSGNSLTEG TLVGLAGLKL PATAGVERDL NGRDYTVTGG FTGATAATG< TGARNMPHTR FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF-
ESVGSISFPE
AVIETEAKPT
NTNDETKNEM
SEEQYRQALL
DAQAQPNTYA
YGSNHCGITA
QKYPWMSNDN
GDFTADTKGT
GGSLVLYGNN
ETVHIKGSLQ
STG1RRVPFLS
YVRRGNAART
TVETAAAflRT
STAAHADMQG
GSTQTVGIAA
AGDIGYLKGL
FAATGDLTVE
SQPLSDYKAVL
LVAGLGADVE
AG983 was expressed as a hybrid, with 0RF46.1, 741, 961 or 961lc at its C-terminus: A&G983-0RF46.1
ATATCGGCCATCAGAGGTCGTTGCGACGAACAACGGATAC
GCAGTATCTTACGCCGGTATCAAGAIACGAATGTGCAAAGACAGAAGCATGCTCTGTcCGGTCGGGATGACGTTGCG
GTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTCCAAACCCAAATGACGCA
TACAAGAATTGATCAACCTCAACCTGCAATGAGCAGGCTATACGGACGCGGGGTAGA.GTAGTATCGTcGAc
ACGCATCTGCGAACTTCCACGAGCGAAACCGTTAG~ATCA
AATTCGGAAGGAGAGGCGAAGGGCGAAAATAGTCTCAGTA
GCGTTGGCGACAGCAGAACGCAGAAGATGAAACATGTTCA
WO 01/64920 PCTLBO1/00420 -14-
ATTATTGGCGGGCGTTCCGTGGACGGCAGACCGCAGGCGGTATTGCGCCCGATGCGACGCTACACATATGAJATAC
AAGTALCAGAGATAGTGACACGATCTGTAGTGCACTGGGG
ATGCAACGTTGAACTGGGAGACGCACTTCATGCATGAGGA
TACCAGGTCCATTCGCGGTAACGCAGTTCCTAGACGGdTA r.CACGCTCAACGATAACTCTTACTTGCGCAGCCCACCGC AAAAAGCTTGCTTATAAAA CCAA~CTATAATGAGGAACCG
GGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTTGGCTCCAACCATTGGGAATT
ACGCTTGGCGCGACTTALCACTCTTACGAAACGTCATGCG
ACTCTTCCCCTGACGCCGGCCTCGTCGATCCTGTACAGCA
CTCTCAGTCGCAGCCGAACGGATGCTGCGAGTGCGG.CGT
GATGCGGGTAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTACCGCCGATACGGTACTCCGAT
ATGCATCTCTAGCTTAGAGGGCTACAALGCGACACGACGA
GGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATG
CGGCAACAGTCCGTTTAGGCGACGCGACTACGGCGATTTT
CTGAAACACACGCCACAACTCAAC;AGATTCGTGCGAAG;C
CTTCrAGTGGAATCGAGGAGTCGGTACGGCAGTTCTTGCCC
AGGGAGTTTCAATCGAGCTGTCTCTATCGCAATGGAGTA
TCTC9CCACTGACGCGGCTCGCTCTGCGGCAAACGGGATA
GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTG.CTTCGGCAGCGGCACATTCCGCGCCCGCC
GGCOALAGCTGAAGCGACACGAACTAGTGATGTCTCATAC
GCAACACCCGAGACGGTTGAACTGCGGCAGCCGACCGCACAGATATCGGGCATCCGCCCCTACCGCTT
CGCGCAGCGGCAGCCGTACAGCATGCGATCCGCCGACGGTGTACGCATCTTCACAGTCTCGCCGCTACCGTCTAT
GCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGGCCGTATCGGACGGGTTGACCCC
ACGTTCCTACCCAACACGAGTGAGGG
AAGCGGTAGCAAGG
GGATCCACGCGATCGGAACGGAATCAACGCCAATGCTGAG
AGCACATGGAGCGAACAGTGCAATGCAACCGACAGCATTAGTCTGTTTCAGGCATACGGCACGATGCGGGC
GATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAACAGCATCAGCCGCAGCACCGQ'pQCGGACGAA
CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCACTGGGCGCACTGGGCGGTGTAACGTTCCGTTTGCCGCAJCG
C-AATGCGCAGCGCGGTCACGTAAAGTCTCCGAAGCGGTT
GGTGGGCAACTATAGCCCGTGGCCCGTTAGTTGACCTACA
AAAGCCGTCCTGTTGCACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACC
GGCGCGACTCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGAT
GTGATGCAGCGACGTGCCTAAGTCCGTCAAATCGACAACG
CGAGTCGGCGTAGGCTACCGGTTCCTCGACGGTGGCGGGCACTGGATCCTCAGATTTGCACGATTCTTTTATC
CGCGTCCACTACTTGACGCGAATCACATGCGAGGGATGCA
CGACGCTTGATGAAAAALGCTCGTGCACG~.TCAAGGCATA
GGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAG'rCCATTCCCCCTTCGACAACCATGCCTCACAT TCCGATTCTGATGAAGCCGGTAI3TCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAiACACCAT
CCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAJGCGCGAGGGATATATACAGCTACGAC
ATAAGGTCCAAACGCCACGCGAACGACCGAACGTGC~CTT
CACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAJACGCGCCACCCGATACAGCCCCGAGCTGGAC
AGTGGATCGCAGCTACGATCGAACTAAAACTGCCGAGGAT
GTGCCGCAGCTCGGAAGGAGCCACTGTTTCCGTGTTCTC
ACGAZAAAGCCCTACATGCGTTGGACCAGCAGCCGACAC~
GATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATC
AAGGGATGGAGCTGTCGGGGAAAATACGGCTTGGGCGGCATCACGGACATCCTTCAAGCGGTCGCAGATGGGC
GCGATCGCATTGCCGAAGGGAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCT
TACTCCAAACGTAATGACGGTCGA-GAAACCTCCACTCGC
TCACGAAAGCACGCGCACCACCAGCGCTCGTGCGAAGTTC
AATTTTGAGAACACGTGAAATAGATACGCTCGAGCACCACCACCACCACCACTGA
1 MTSAPDFNAG GTGIGSNSRA TITAKSAAVSY AGIKNEMCKD RSNLCAGRfDD 51 VAVTDIRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV 101 GIVDTGESVG SISFPELYGR KEHGYN'ENYK NYTAYMRKEA PEDGGGKDIE 151 ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIGGRSVD GRPAGGIAPD 201 ATLHIMNTND ETKNMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD 251 LFQIMSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLiS YHINNMF 301 IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KPKREMYGEP 351 GTEPLEYGSN HCGITAMWCL BAPYEASVRF TRTNPIQIAG TSFSAPIVTG 401 TAMLLQKYP WMSNDNLRTT LLTTAQDIGA VMVSKPGWG LLDAGKAMG 451 PASFPFGDPT AflTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT 501 GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIV'iLAflT 551 DQSGAN'E1'VH U(GSLQLDGK GTLYTRLGKL LKVDGTAIG. GLYMSARGK 601 GAGYLNSTGR RVPFLSAAXI GQDYSFTNI ETDGGLLIASL DSVEKTAGSE 651 GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NL14VELDASE 701 SSATPETVET AAAflRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA 751 ATVYADSTAA HAflMQGRRLK AVSDGLDHNGTGLRVIAQTQ QDGGTWEQGG WO 01/64920 PCT/IBO1/00420 801 VEGIKMGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF 851 AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADFlaUGSV NGThMQLGAL 901 GGVKVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG 951 LAGLKLSQPL SDKAVLFATA GVERDI.NGRfl YTVTGGFTGA TAATGKTGAR 1001 NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLD 1051 GGGGTGSSDL ANDSFIRQVL DRQHFEPDGI( YHLFGSRGEL AERSGHIGLG 1101 KIEQSHQLGNt MIEQQAAIKGN IGYIVRFSDH GHEVHSPFDN HASHSDSDEA 1151 GSPVDGF~SLY RIHWDOYEHH PAflGYflGPQG GGYPAPKGAR DIYSYDIKGV 1201 AQNIRLNLTD NRSTGQRLAfl RFHNAGSMLT QGVGDGFKA TRYSPELDRS 1251 GNAAEAPNGT ADfIVKNIIGA AGEIVGAGDA VQGISEGSNI AV1MHGLGLLS 1301 TENR1MRID LAflMAOLKDY AAAAIRDWAV QNPNAAQGIE AVSNIFMAAI 1351 PIKGIGAVRG KYGLGGITAH PIKRSQMGAI ALPKGKSAVS DNFADAAYAK 1401 YPSPYHSRNI RSNLEQRYGK ENITSSTVPP SNGEJPIKAfl QRHPKTGVPF 1451 DGKGFPNFEK HVKlYDTLEHHS HHHH~* AG9 83-741
ATATCGGCGCTATCGCGACGATGCGACGAACAACGGATAC
GCGACTCCGTTAGAGATTCAGAAAGAGTTTCGTGGTAGTC
GTAAAAGAGCAACAGCCCCCAACGAACGGCTCAACAAGCC
TACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGAC
ACGCATCTGCGAACTTCGATTAGCGAAACCGTTAGAATCA
AATTCGGAAGGAGAGGCGAAGGGCGAAAATAGTCTCAGTA
GCCGTTATAGAGACTGAAGCAAGCCGACGGATATCCGCACGTAGATCGACACATCGATTTGTCTCCAT
ATATGGGGTCTGCGAACGAGGGATCCCAGGCCAAAATATC
AAGTAACAACAAGTGTCGCTCGATCTGTAGTGCACTGGGG
ATGCAACGTTGAACTGGGAGACGCACTTCATGCATGAGGA
TACCAGGTCCATTCGCGGTAACGCAGTTCCTAGACGGGTA
GGACTTCACCTCTAAAAAGTTTACTTGCGCAGCCCACCGC
AACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGT
GGGALGTAAGGATTTGGACGTCGACCTATTGTCACTGGAT
ACGCTTGGCGCGACTTAGAGGTCTTACGAAACGTCATGCG
ACTCTTCCCCTGACGCCGGCCTCGTCGATCCTGTACAGCA
CTCTCAGTCGCAGCCGAACGGCGCGGGAACATCGTGGCGT
GATGCGGGTAAGGCCATACGGACCCGCGTCCTTTCCGTTCGGCGACr'rACGCCGATACGAAGGTACATCCGAT
ATGCATCTCTAGCTTAGAGGGCTACAAAGCGACALTCA~.A
GGACAACAAGGAACATTGAGGGTGTGGTTCGACAAACGTT
CGCGTCGAAACCAGGTGCGCTGATTTATAACGGCATCCGGCGAGCCCGCGACGATTTCTAT
CTGAAACACACGCCAAGACGAAACAGCGCGACGAGCAGTC
CTTCCCTTGCACGTAATGCGACGGTACGGCACGAAGCGAG
GGCAAGGGGGCAGGCTATCTCACAGTACGGACACGTGTTCCCTTCCTGAGTGCCGCACGGCGGATA
TCTCTAAAACAACACGGCTCGGTCCc~AcTGAA.CGGGATA
GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCC
GGCGACCCGAACGGGCGATTGAACTAGTGATGTCTCATAC
GCAACGGCGTBkCGGCGCACCCGTTCGGACGCCAGCCATT
CGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAAOAGTCTCGCCGCTACCGTCTAT
GCCGACAGTACCGCCGCCCATGCCGATATpGCAGGGACGCCGCCTGAGCCGTATCGGACGGGTTGGACCACAJACGGC
ACGTTCCTACCCACCAAGCGGACTGACGGGTTG.GCAAGG
GGATCCACGCGATCGGAACGGAATCAACGCCAATGCTGAG
AGAAGACAACGGAAGAAACAAGATGCGTGAGAAGCCAGGG
GAACGTTTAAGCGTTCAGAGTAAAAACTACGACCGTCGCA
CAGGAGCGGCAGCCCGTCGTGGGATGCGGCAGTCTTCGAC
GGGTTAGTGAGGTTCCAGCTCTAAAGTCTCCGAAGCGGTT
GGTGGGCAACTATAGCCCGTGGCCCGTTAGTTGACCTACA
AALAGCCGTCCTGTTTGCAACGGCGGGCGTGGACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACC
GGGGCGACACGAGCGGCCCAAGCCCCCTTGTCGCTGCGGA
GTCATCGACGTGAGCTGAGTCACAGCGTCACGAGCCAACG
CGGCGGAGTCGTCTGGGTCGGWTGGCCGCAACGGGGTC
GAGATACCCGTGCAAAGCAGTTCGCTGCCGACGCGCGAAA
GAAATAGTGGCCAGGGAAACTAGAAGTAACTATCGCATGA
AACCAGCGCTTGCTACGCATGAGGAGOACCTACTGGGGAA
TTCATTCACAGCTCGCTACCTTCGCGGAAAAGTCGGATCG
AAAGTGGACCATCGACGGCTGCGCACTCTTTGCACTCGAG
GGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCG3AAAJACTGACCTACACCATAGATTTCGCC
GCAGAG-AGCAACACTTAACCCGATATTGCTGCCGCAACA
CCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAA.CCAAGCGAlAAGGCAGTTACTCCCTCGGT WO 01/64920 PCTIBO1/00420 -16-
ATTTGGAAGCAGATGCGACCGAGGAACTACGAAGCTTGCT
GCCGCCAAGCAACTCGAGCACCACCACCACACCACTGA
1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 9 51 1001 1051 1101 1151 1201 1251 1301 MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKEMdCKD.RSIMLCAGRDD VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV GIVDTGESVG SISFPELYGR KEHGYNE1NYX N.YT1AYMRI(EA PEDGGGKDIE ASPDDEAVIE, TEAXPTD:IRH VXEIGHIDLV SHIXGGRSVD GRPAGGIAPD ATLHIMNTUD ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD LFQIMSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHflRNKNMLF IFSTGNDAQA QPNTYALLPF YEKDAQ1KGII TVAGVDRSGE KFKREMYGEP GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG.TSFSAPIVTG TAALiLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKMNG PASFPFGDPT A.fTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT GKTIUEGGSL VLYGNINK5DM RVETKGALIY NGAASGGSLN SDGIVYLADT DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GFLYMSARGK GAGYIJNSTGR RVPFLSA?.KI GQDYSFP1'NI ETDGGLLASL DSVEKTAGSE GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRI:FNSLA ATVYADSTAA }HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG VEGXGIRGSTQ TVGIAAXTGE NTTAAATLGI4 GRSTWSENSA NAI(TDSISLF AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMLGAL GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLT9EGTLVG LAGIACLSQPL SDKAVLFATA GVERDLNGRD YTVTGGPTGA TAATGKTGAR N"~HTRLVAG LGADVF9GNG tWNGIJARYSYA GSKQ-YGNHSG RVGVGY1RFLE GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKN KVSRPDFIRQ IEVDGQLITL ESG9EFQVYKQ SHSALTAPQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT YRGTAPGSDD AGGKCLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD GKFRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL AAKQLEHHHH HH* AG9893-961
ATATCGGCGCTATCGCGACGATGCGACGAACAACGGATAC
C-ATTTAGCGACAACAAGGAAAAAGAGTTTCGTGGTAGTC
GTTACAGACAGGGATGCCAAAATAATGCCCCCCCCCCGATCTGATACCGGAGACTTTC AACCCA TGCGCA
TAAGATGTACTAACGATGACGGTTCGAGGGTGGTGTTGCA
ACGCATCTGCGAACTTCGATTAGCGAAACCGTTAGAATCA
AACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGA
GGCGGTAAGCTGATTTTCGACGATGAG
GCGTTGGCGACAGCAGAACGCAGAAGATGAAACATGTTCA
ATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCGCGGTATTGCGCCCATGCGACGCTACACAATGATACG
AAGTAACAACALTAGTGACACCATCTGTAGTGCACTGGGG
ATGCAACGTTGAACTGGGAGACGCACTTCRTGCATGAGGA
TACCAGGTCCATTCGCGGTAACGCAGTTCCTAGACGGGTA
GOACTTCACCTCTAAAAAGTTTACTTGCGCAGCCCACCGC
AAAAAGCTTGCTTAGAAGCCCAACGATTAATGAGGAACCG
GGGAATCACGAAGAGAACGGACGACCTATTGTCACTGGAT
ACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGrCGTCCGTTTCACCCGTACCCGAAATTGCGGA
ACTCTTCCCCTGACGCCGGCCTCGTCGATCCTGTACAGCA
CTCTCAGTCGCAGCCGAACGGCGCGGGAACATCGTGGCGT
GATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTACCCGATACGJLAGGTACTCCGAT
ATGCATCTCTAGATCGCCGCGCGTAAAGCGACACGACGA
GGACAACAAGGAACATTGAGGGTGTGGTTCGACAAACGTT
CGGCAACAGTCCGTTTAGGCGCTCGGCGCGAACAGCTGCA
CTGAAACACACGCCACAACTCCTAAGATTCGTGCGA6GTC
CTGTACACACGTTTGGGCAAACTGCTGAGTGGACGGTACGGCGATTATCGGCGGAGCTGTACTGTGGCACGC
GGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTTGCCGCATCGGGAGGATTAT
TCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGAC GCGTCr.AAAA~,GAGG
GGGCCCGCTTAGCGCCGATCGAGA~.TCGACGAATCCCCC
GGCGACCCGAACCGGVA-AP.(
WO 01/64920 WO 0164920PCT/IBO1/00420 -17-
CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACG.
GGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAGATGCATTCGCCGAGCAGGCTTG
GGCTGGAGCGGCAACAGCCTCACTGAGGOCGCTGGTCGGACTCGCGGGTCTGAAGCTrGTCGCACCCTTGAGCCAT
AAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCACCGAACGGACGCGACTAACGGTACGGGCGGCTTTACC
GTCGAATTCGGCAACGGCTGGACGGCTTGGCACGTACAGCACGCCGGTTCCAACAGTACGGCAACCACAGCGGA
CGGCGGAGTCGTCTGGGGCGGGATGTCCAAAGCAGTTAAA
GCTGCCACTGTGGCCATGCTGCTGCCTACAACAATGGCCAAGAATCAACGGTTCAAGCTGGAGAGACCATCTAC
GAATAGAAGCCATCAAAGCCACGACGTTGACGCATTAGTT
G.GTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAACAAIACAAACGTCGATGCCAAAGTAAAAGcT
GCAGAATCTGAAAAGAAAAGTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTIGATGCCGCTCTG
GATGCAACCACCAACGCCTTGTATTGGGAGAATATAACGACATTGCTGQGAGACTAGACAATATCGTA
AAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCA
TTGTAACACCAGCrAGACGCAACCATAGCACGCGCAGAACA CAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAGCCGAAGCTGCCGCTGGCACAG3CTAATACT GCGCAAGCGACGCCGAAGTCGCTAACGTT CAGAAAAATT
GCAAAGAAATCGCTTCCAAAGATTAACATTTAATGTGCGA
GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTG.CTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGT
TTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTC
CAACCTTAAACGTGGGTCGGTTCATGTAACGGCTGCAGTCGGCGGCTACCACGGCAGTCGCCATCGGT
ACGCTCCTACA~CTGCCAACGCTG.GCGATCTCrTTCGAC
TACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA
1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 MTSAPDFNAG GTGIGSNSRA TTAXSAAVSY AGIKNEMCKD RS1ILCAGRtD VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV GIVDTGESVG SISFPELYGR KEHGYNENYI( NYTAYMR(EA PEDGGGHDIE ASFDDEAVIE TEAKPTDIRH VKEI1GHIDLV SHIIGGRSVD GRPAGGIAPD ATLHIMNTND ETKNEM14VAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAfl LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHI1NKNMLF IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFXREKYGEP GTEPLEYGSN HCGITA4WCL SAPYEASVRF TRTNPIQIAG TSFSAPflhTG TAALLLQ8ZYP WMSNDNLRTT LIJTTAQDIGA VGVDSKFGWG LLDAGKAMG PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQIMGNNTYT GKTIIEGGSL VLYGNNI(SDM RVETKGALIY NGAASGGSLN SDGIVYLAflT DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK GAGYLNSTGR RVPFLSAAI(I GQDYSFFTNI ETDGGLLASL DSVEKTAGSB GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHAINAA DGVRIFNSLA ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG VEGKMRGS'rQ TVGIAARTGE NTTAAATLGM GRSTWSEN'SA NAKTDSISLF AGIRHDAGDI GYLKGLFSYG RYRNSISRBT GAflEHAEGSV NGTLMQLGAL GGVNVPFAAT GDLTVEGGLR YDLLKQD1AFA EKGSALOWSG NSLTECTLVG LAGLKU.SQPL SDI(AVLFATA GVERflLNGRD YTVTGGFTGA TAATGKTGAR I'ThPHTRLVAG LGAflVEFG.NG WNGLARYSYA GSKQYGNHSG RVGVGYRFIJE GGGGTGSATN DDDVKKAATV AIAAAYNNGQ BINGFKAGET IYDIDEDGTI TKKDATAAflV EAflDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE KLTTICAflTD AALAflTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE KLEAVADTVD KHA.MMNIA DSLDETNTKA DEAVE'rANEA KQTAEETKQN VDAKVKAAET AAGKAEAAAG TA1NTAADKAE AVAAKVTDII( ADIATNKDNI AKKANSADVY TREESDSKFV RIDGLNATTIE KLDTRLASAE KSIADHDTRL NGLDKTVSDL RKETRQGL1AE QAALSGLFQP.YNVGRFNVTA AVGGYKSESA VAIGTGFRF'T ENFAACAGVA VGTSSGSSAA YHVGVNYEWL EHHHHH* AG983-961c
ATATCGGCGCTATCGCGACGATGCGACGAACAACGGATAC
GCGACTCCGTTAGAGAAGGAAAAAGAGTTTCGTGGTAGTC
GTAAAAGAGCAACAGCCCCCATTCTCWGCWCACCATAW
TAAGATGTACTAACGATGACGGTTCGAGGGTGGTGTTGCA
ACGCATCTGCGAACTTCGATTAGCGAAACCGTTAGAATCA
AATTCGGAAGGAGAGGCGAAGGGCGAAAATAGTCTCAGTA
GCCGTTATAGAGACTAAGCGCGACGGATATCCGCCACGTATGGACCAGATTTGGTCTCCCAT
ATATGGGGTCTGCGAACGAGGGATCCCAGGCCAAAATATC
AAGTAACAACAA~TTGACACGATCTGTAGTGCACTGGGG
ATCGTCAATAACtTTTTGGAACAACATCGGGGGCACTGCCGACCTTTTCCAATAGCCAATTCGGGGAGCAG
TACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAGACG;AGGGTATCCGCTATGCACAAGCATTAC
GOACTTCACCTCTAAALCTCTTACTTGCGCAGCCCACCGC
WO 01/64920 PCT/TIBO1/00420 AAACTAGCCTTTCCTTTAGAAAAGCGTAAAAGCTTTCCATCCAG-18-CGCG
GGGAATCACG-ATTTGGACGTCGACCTATTGTCACTGGAT
ACGCTTGGCGCGACTTAGAGGTCTTACGAAACGTCATGCG
ACTCTTCCCCTGACGCCGGCCGTCGAAAACGGAGGACA.CA
TCTCAGTCGCAGCCGAACGTCGCGGGAACATCGTGGCGT
GGGAGCAGACGCCGCTTCTTGCATTCGCATCA~.T~TCA
ATGCATCTCTAGCTTAGAGGCGCGTAAAGCGACACG
CGA
GGACAACAAGGAACATTGAGGGTGTGGTTCGACAAACGTT
CGrTGACAAGTCCGTTTAGGC.GACGC-CGCCUACAGCTTTT
TCTTTCTTCACAAACATCGAAACCACGGCGGCCTGCTGGCTTCCCTCGACAGGJACGCGGGCAGTGAA
GGGCCCGCTTAGCGCCGATCGCCGCGTCGAC CrATCCCCC
GGCGAAAGCTGAAGCGACACGGAACGTGC;ATGTCTCATAC
GCAACGGCGTAATCGACGCGACGTTCGGACGCCAGCCATT
CGGACGACGAACTCATCGCAGGGAGACTACGCCCGTCGCA
GCGCGACCGCAGCAAGAGAGCGCGAGCTTGAGGTGCAACG
ACGTTCCTACCCACCAAGCGGGAGGGAAGCGGTAGCAAGG
GGATCCACGCGATCGGAACGGAATCAACGCCAATGCTGAG
AGAA~-GGAAATCATCAACGCGATGCGT~A~AAGCCAGGG
GAACGTTTAAGCGTTCAGAGT.AAAACTACGACCGTCGCA
CAGGAGCGGCAGCCCGTCGTGGGATGCGGCAGTCTTCGAC
GGGTTAGTGAGGTTCCAGCTCTAAAGTCTCCGAAGCGGTT
C-CGACGACGCCCGAGAGTGCGCCCGTTAGTTGACCTACA
AAGCTCGTGACGGGGGACCACTACGCCATCCGACGCGTTC
GGGGCGACACGAGCGGCCCAATCGAACGCGTGCGCGGGGA
GTGATGCAGCGACGTGCCTAAGTCCGT6AAATCGACAACG
CGGCGGAGTCGTCTGGGGCGGGATGTCCAAAGCAGTTAAA
GCGCCGGCATCGTCTCAATGCAGATACGTCAGTGGGCACA
GAATAGAAGCCATCAAAGCCACGACGTTGACGCATTAGTT
GGCGAAATGGCACTACAACTAAGAAAAAACTGTCAATAAC
GCGACGATGAATACACATACGAATAGCCTACGTCGTCGTT
GAGACACAGCTATATCWGAAAACAAITTCGAAATAAAAACT
AAATAGAATAAGCTGTAACTGAAGAGCAGATACAACCGTC
TTGT-ACACCAGCGCAGCTAACGCAGAGCACGCGCAAACA
CAACTGTCAATAAcGAAATCGCGCAGCAGTCGTGAACATC GCrCGCAGCAGTTGTCAATACAAcA~TAACCAGAAAAATT GCAAAGAAATCGCTTCCAAAGGCGCGAATGCGArGTGCGA
GCATCGAATGAAAGTGCTTCGA-.ACATCGTAGTCCCTACG
TTGTAAATTAACGGAAAACGCAGCTGAACACGGTTCGCGT
CACTAACTGTTGGACCACCACCG
1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 MTSAPDB'NAG GTGIGSNSRA TTAI(SAAVSY AGI:KNEMCRKD RSMLCAGRDD VAVTDRDAKI NAPPPNLIITG DPPNPNDAYK INhINLKPAIE AGYTGRGVEV GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA.PEDGGGKDIE ASFDDEAVIE TEAKPTDIRH VXEIGHIDLV SHIIGGRSVD GRPAGGIAPD ATLHIMNTND ETIQIEMVAA IRNAWVYLGE RGVRIVNNSF GTTSRAGTAfl LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRKNNLF IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKMM14GEP GTEPLEYGSN HCGIAMWCL SAPYF.AS'.mF TRTNPIQIAG TSFSAPIVTG TAALLLQKYP WXSNDNLRTT LLTTAQDIGA VGVDSKFGWG.LLDAGKMNG PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT GKTIIEGGSL VLYGNNKSDM RVETKGAI.IY NGAASGGSLNq SDGIVYLAflT DQSGANETVH IKGSLQLDGI( GTLYTRLGKL LKVDGTAIXG GKLYMSARGK GAGYLNSTGR HVPFLSAAXI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE GDTLS'iYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE SSATPETVET AAAflRTDNPG IRPYGATFRA AAAVOHANAA DGVRIFNSSLA ATVYAflSTAA HADXQGRRIA( AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSBNSA NAKTDSISLP' AGIRHDAGDI GYLKGLFSYG RYKNSISRST GAflEHAEGSV NGTLXQLGAL GGVNVPFAAT GDLTVEGGLR YDLLKQDAPA EKGSALGWSG NSLTEGTLVG LAGLRLSQPL SDKAVLFATA GVERDLNGRD YTVTGGP'TGA 'rAATM=TAR NMPHTRLVAG LGAflVBPGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE GGGGTGSATN DDDVKXAATV AIAAAYNNGO BflNGFKAGET IYDIDEDGTI TKKDATAAflV EAflDFKGLGL RKVVTNLTKT VNENXQNVDA KVKAAESEIE KLT'rKLADTD AALADTDA?.L IDATTNALNKL GENITTFABE TrTNivxKIDB WO 01/64920 PCTLBO1/00420 -19- 1.201 KLEAVADTVD KHAEMMWIA DSLDETNTKA DEAVI(TANEA I(QTAEETXQN 1251 VDAI(VKAAET AAGRAEAAAG3 TANTAhDKAE AVAA2KVTDIX ADIATNKDNI 1301 A1K(ANSADVY TREESDSKFV RIDGLjNATTE KLDTRLASAE XSIAflHDTRL 1351 NGLDKTVSDL RI(ETRQGLAE QAALSGLFQP YNVGLEHIIH HH* Example 4 -hIybrids of AG741 'Protein 741 has the following sequence:, 1 VNRTAPCCLS LTTALILTAC SSGGGGVAAfl IGAGLADALT APLDHKDKGL 51 QSLTIDQSVR KNBKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ 101 IEVDGQLITL ESGEPQVYI(Q SHSALTABQT EQIQDSEHSG OWAXRQFRI 151 GDIAGEHTSF DKLPEGGRAT YRGTAPGSDD AGGKLTYTID FAAKQGNGKI 201 EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEXGS YSLGIFGGKA 251 QEVAGSAEVI, TVNGIRHIGL AAXQ* AG741 thus has the following basic sequence: VAAD IGAGLADALT APLDHKKGL QSLTLDQSVR KlqEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEQVYKQ SHSALTAFQT EQIQDSEHSG KRVAM~QFRI GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAICQGNGKI EHLKSPELNV DLAAADIPXPD GKRHAVISGS VLYNQAflKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL AAKQ* AG741 was fused directly in-frame upstream of proteins 961, 96 Ic, 983 and 0RF46. 1: AG742.-961 ATGCCGCAACGGGGCTCGTCCTACCCG~rACTAGCAGTTCL
TCTGCCGACGCGCGAAAGGACGACGCGAAGGGAAACTTG
AACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAATCGAAGTG
GACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTccAAGTATAcAAcACAGcATTccGccTTAACCGCdTTTCAG
ACGGAAAAGTCGGATCGAGTGTGGACCATCGACGGCTGGG
GAACATACATCTTTTACAAGCTTCCCGAAGCGGCAGGGCGACATACGCGGGACGGCGTTCGGTTCAGCGATGCC
GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA
CTCAATGTCGACCTGGCCGCCGCCGA'rATCAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCICTTTAC
AACCAAGCCGAAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAGCCCAGGAGTTGCCGCAGCGCGAGTG
AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGTGGCGGAGGCACTGGIATCCGCCACA
AACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACATGGCCAAGAAATCAACGGTTTC
AAGTGGGCACAGCTGTAGCGAATACA;AGCCATCGCAGTA
GCGCATTAGTTGTT-AAATGGCACTACAACTATA&CACAA
GTCGATGCCAAGTAAAAGCTGCAGAATCTGAAATAGAAAGTTAACAACCAAGTTAGCAc3ACACTGATGCCGCTTTA
GCGTCGTCGTTGTCACCACCTTATATGGGAAAACAATGTA
GAAThAAAACTAATGTAAATGACCTGTAACTGCACTCGAC TTACAACCGTCTGAGACACCAGCGCAGCTAA~rcATA~cA
CAGACGGCCGAAGAAACCAACAAAACGTCGATGCCAAAGTAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCT
GCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAGTTACCGACATCAAAGCTGATATC
GCTACGAACAAAGATAATATTGCTAAAAGCAAIACAGTGCCGACGTGTACACCAGGAAGAGTCTGACAGCAALATTT
GTCAGAATTGATGGTCTGAACGCTACTACCGAAAAJATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGAT
CACGATACTGCCTGACGGTTTGGATAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAA
GCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCATGTAACGGCTGCAGTCGGCGGCTACAAATCC
GAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGACTTTGCGCAGCAGCTGGCATCGGCACT
TCGTCCGGTTC'rTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA 1 XVA2W1.GACL ADALTAPLDH KDRGLQSLTL DQSVRIMMKL KLAAQGAEKT 51 YGNGDSLNTG 1(LKNDKVSRF DFIRQIEVDG QLITLESGEB' QVYKQSHSAL 101 TAFQTEQIQD SEESGKMVA1( RQFRIGDIAG EHTSFDI(LPE GGRATYRGTA 151 FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA 201 VISGSVLYNQ AEKGSYSLGI FGGKKQEVAG SAEVKITVI'GI 1RHIGLAA(QL 251 EGGGGTGSAT NDDDVKKCAAT VAIAAAYNNG QEINGKAGB TMYIDEDGT 301 ITKKDATAAD VEADDFKGLG L RVVNLTI( TVNENKQN.VD AKKAESEI 351 ERLTTKLADT DAALADTDAA LDATTXAL*K LGENITTPAE ETKTNIVKID 401 EKLEAVADTV DIMIAEAFPqI ?.DSLDETNTK ADAVITAXE AK~QTAEETKQ WO 01/64920 PCT/LEBO1/00420 451 NVDAXVKAAE TAAGXAEAAA GTANTAAflRA EAVAA(VTDI KAflIATNqKDN 501 IAKKANSADV YTREESDSKF VRIDGLNAT' EKLDTRLASA EKSIAflHDTR 551 LNGIJDKTVSD LPRKTRQGLA EQAALSGLFQ PYNVGRFNVT AAVGGYICSES 601 AVAIGTOFEF TENPAAKAGV AVGTSSGSSA AYHVGVNnE w LEHHHHH* A0741-961c ATGCCGCAACGGGGCTCGTCCTAccCGTGCAAAAAAGTGA
TCTGCCGACGCGCGAAAGGACTAGTGGCCAGGGAAACTTG
AAGTAACTATCGCATGAGAGCAGCGCTTGCTACGCATGAT
GAGGACCTACTGGGGAATCAGAAAACAGCTCGCTACCTTA
ACGGAAAAGTCGGATCGAGTGTGGACCATCGACGGCTGGG
GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCACGCGTCGGTGACGATCC
GrCGAATACAACTGTTGCCAGCGGACrCAACACTTAACCAA
CTATTGCTG~.CCGTTAGCGTGAAAGCTCGCTACGTCTCTA
AACA-CAAAGATATCTGTTTTGGA"CAGATGCGACCGAT
AAACTACGAAGCTTGCTGCCCACATGGGGrGCCGTCC~-
AAGCAGTTAAAG~.CCGGCATCGTCTCAATGCAAACAGT~
AAGTGGG~Ac~G~ATAGA~Gc~AT~cAAA~~Ac~AcG~.TA
GCGCATTAGTTGTTAAAGCTACACTACAACTATAACACAA
GTGIALGAA(CG~-ACGATGAATACACATACGCCGTCGTT
GCGTCGTCGTTGTCACCACCTTATATGGGAAAACAATGTA
GAATAAAAA~-AAATAGAATAAGCTGTAACTGCACTCGAC
TTACAACCGTCTGAGAcACCAAGAAGACGCAAC~ATAGCA CAAGCGAAACACA~GcAGCAGTAACGAAATrGAGAACGAC GCCGCTGGCACAGCTAATACTGCAGCCGAAGGCCGA
TGTCGAGACCGAATAAGTGATATC
r-TCACAGTAATCAAACACGTCGCTTCCAAAGGCGCGAAT
GTAATGTGCGAGTCACAAATGAAAGTGCTTCGAATCTGCA
CAGTCCCTACGT~AAACGGCGACGGAAAACGCAGCTCGAA
GCGGTTCGCGTCACTCAGTGTTGGACCACCACCG
1 MVAAflIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNflKL KLAAQGAEKT 51 YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL 101 TAPQTEQIQD SEHSGQ4VAI( RQFRIGDIAG EHTSFDKLPE GGRATYRGTA 151 FGSDDAGGKL TYTIDFAAXQ GNGKIEHLKS PELNVDLAAA DIEKPDGKRHA 201 VISGSJLYNQ ABEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL 251 EGGGGTGSAT NDDDVKK(AAT VAIAkAAYNNG QEINGFKAGE TIYDIDEDGT 301 ITKKDATAAD VEAflDFKGLG LKKVVTNLTK TvNENKQNvD AKVKAAESEI 351 EKLTTKLADT DAALADTDAA LDATTNALNKC LGENITTFAE ETKTNIVKID) 401 EKLEAVAflTV DKHAEAFNDI AflSLDETNTK AflBAVKTNE AKQTAEETKQ 451 NVflAKVKAAE TAAGHAEAAA GTANTAADKA HAVAAKVTDI KADIATNKDN 501 XAIKANSADV YTREESDSIKF VRIDGLNATT EKLDTRI.ASA EKSI:ADHJTR 551 LNGLDKTVSD LRKMTRQGLA EQAALSGLFQ pyNVGLEHHH HHH* AG741-983 ATGCCGCAACGGGGCTCGTCcTACCCGTGCAAAAAAGTGA
TCTGCCGACGCGCGAAAGGACTAGTGGCCAGGGAAACTTG
AAGTAACTATCGCATGAAC~AAGCGCTTGCTACGCATGAT
GAGGACCTACTGGGCAATCAGTTcACAGCTCGCTACCTTA
ACGGAAAAGTCGOATCGAGTGTGGAGCGTAATGGCTGG~
GACTCTTTGCACTCGAGGCGGCAAACCGAGCTCGTAAGTc
GGGAACGCTCCAAATCCGCACGGACGAATGAATGACCAA
CTATTGCTGCCGCAACAcGAGGAAGCTCGCTACGTCTCTA AACACGGAGCGTCCCCGACTGCGAACCGAGTCGCrCCGAT
AAACTACGAAGCTTGCTGCCAGACCAGTCGCGGCGATCC(
CCGCTATCGCGACGACGACAArAACAACGGATACGATTTA
GCGTTAGAGATTCAGCGAAGTTTCGTGGTAGTCGTCGCG
G;TCAATATCCCCCGACGAACGGCTCAACAAGCCTCAATT
ATACTAACGATGACGCAAAGCGGGTGGTGTTGCAAAGGAC
GTGCGAACTTCGATTTGAAAGACCGTTAGAATCAACAAGC
TAAGGAGArCCTACACGGCGAAGcTGAc~T~GcArA~CTAAA
ACGACAGCAGAACGCCTAAAATGAAACATGTTCAATTGCG
CGTCTGCGAACGAGGTTGGCOTCAGTCCTATCATAGAC
AAACAAGTCTCGCTCCAGAGGTAGTGCACTGGGGACTATA
AGTTG;CAACAGCGCCGCACTTCAAACATC(AGGATCGCAC
TTCCATTCGCGGTAAA~GGGATCCTAGACGGGTAGCACGC
WO 01/64920 PCT/TBOI/00420 -21- TTGCTCGACGCTTCAGGGTCAGCGGGCGGC CAG CGGCGAGCTGCTCGGGAAC
GCAGAGACGGCTTCTCGGCTACGCAAGAGTCTCAATCTCC
TAGCTTCGCCGCGCTAMAAGCGCGCATcATGAGCAACC TAAGGAACATTGAGGTCCGTTGAGCAACATGAAGGClGAC
AAGGGTATAACGGGCACGCGCGCGAACAGCTGCACGCGTC
GACACGCCACAACTCCTAAGAGCGACGAGCAGTCCGAAAG
CCTATATGTCGCGGAATGCG CGCACGACGCTTCGGCAGTCGCCCGCACCCGGTCT~GAAACC GCGAACrGCGACACGA-ACGTGCACGAGCCGACTCCACCCA
TAATCGACCACCCGTTCGGCTCCCTCGGCATTCCCGGC
GCGAACTCATCGCAGGAGACTCAATTGCCACTTTCGCGAC
GCGCAGCAAGAGAGCCTAACGACGCGTGACCACGAGGCGG
GCTGGAACACGAGTGAGGGAAGCGGTAGCAAGGGCGACA
ACGCGATCGGAACGGAAAGCGCGCCAATGCTGAGACCTGG
3 CTAAGCCGTTCTCGAGGACCACACAACACATACGAACGTGAGAAGGAG 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 MVAADIGAGL AflAIAAPLDH KflKGLQSLTL DQSVRKNEKL KLAAQGAEKT YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF' QVYKQSHSAL TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGPJATYRGTA FGSDDAGGKL TYTID1'AAI(Q GNGKIEHLKS pELNVDLAAA DIKPDGK1RRA VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEWKTVNGI RHIGLAAKQL EGSGGGGTSA PDFNAGGTGI GSNSRATTAk SAAVSYAGIK NEMCKDRSML CAGRDfDVAVT D1RDAKINAPP PNLHTGDFPN PNDAYKNLIN LKPAIEA3YT GRGVEVGIVD TGESVGSISF PELYGRKEHG YNENYKNYTA YMRKEAPEDG GGKflIRASFD DEAVIETEAK PTDIRHVEI GHIDLVSHII GGRSVDGRPA GGIAPDATLH fl4NTNDETKN EKVAAIRINA WVKLGERMV IVNNSFGTTS RAGTADLFQI ANSBEQYRQA LLDYSGGDKT DEGIRI.MQQS DYGNLSYHIR NKNMLFIFST GNDAQAQPNT YALLPPYEAW AQKGIITVAG VDRSGEKFKR EXYGEPGTEP LEYGSNHCGI TAMWCLSAPY EASVRPTRTN PIQIAGTSFS APIVTGTAAL LLQKYPWMSN DNLRTTLLTT AQDIGAVGVD SKFGWGLLDA GKAbdNGPASF PFGDFTADTK GTSDIAYSFR N'DISGTGGLI KKGGSQLQI.LH GNNTYTGKTI IEGGSLVLYG NNIKSDMRVET KGALIYNGAA SGGSLNSDGI VYLADT'DQSG AINETVHIKGS LQLDGKGTLY TRLGKLLKVD GTAIIGGKLY 1MSARGKGAGY LNSTGPRRVPF LSAAKIGQDY SFPTNIETDG GLLASLDSVE I(TAGSEGDTL SYYVRRGNAA RTASAAAHSA PAGLKHAVEQ GGSNLENLM4V ELDASESSAT PETVETAAAD RTD1MPGIRPY GATFRAAAAV QHANAADGVfl IFNSLAATVY AflSTAAHAflM QGRRLKAVSD GLDHNGTGLR VIAQTQQDGG TWEQGGVEGX MRGSTQ'IGI AAKTGENTTA AATLGMGRST WSENSANAI(T DSISLFAGIR HDAGDIGYLK GLPSYGRYKN SISRSTGAflE HAEGSVNGTL MQLGALGGVN VPFAATGDLT VEGGLRYDLL KQDAFAEIKGS ALGWSGNSLT EGTLVGLAGL KLSQPLSDKA VLiPATAGVER.DLNGRDYTVT GGFTGATAAT GKTGAH1NMPH TRLVAGLGAD VEFGNGWNGL ARYSYAGSCQ YGNHSGRVGV GYRPLEHHfHH HH* AG741-0EP46. 1
ATGCCGCAACGGGGCTCGTCCTACCCGTGCAAAAAAGTGA
TCTGCCGACGCGCGAAAGGAcTA~T~GccAG~GAAAcTTG
AAGT~ACTATCGCATGAACAAAGCGCTTGCTACGCATGAT
GAGGACCTACTGGGGAATCAGTTCACAACATCCTACGCTCr
ACCAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAACGCCAGTTCAGAATCGGCGACATACGGGC
CACAACTTAAGTCCAGCGAGCAAACCGAGCTCGTAAGTC
IWO 01/64920 PCTLBO1/00420 -22-
GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA
CTfCAWI'GTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTAC
AACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTGCCGGCAGCGCGGAAGTG
AAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCACGGrGGCGGAGcACTGGATCCTcAGAT TTGGCAAACGATTCTT'rTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGC AGCAGGGGGGAACTT1GCCGAGCGCAGCGGCCATATCGGATTGGCGAAAATACAAAGCCATCAGTTGGGCAACCTGATG AT1'CAACAG3GCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCC
TTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCG;TTGACGGATTTAGCCTTTACCGCATCCAT
TGGGACGGATACGAACACCATCCCGCCGAcGGcTATG.ACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCG AGGGATATATACAGCTACGACATAAAAGGTTrGCccAAAATATCCGcCTC-AAcCTGACCGACAACCGCAGCACCGGA
CAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGATAGGCGACGGATTCAAACGCGCCACC
CGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCAC'rGCAGATATCGTTAAAAACATC
ATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATG
CACGGCTTr.GGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGAC TATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATA3AAGCCGTCAGCAATATC
TTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCT
ATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAA'rTTTGCCGATGCGGCA
TACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGCAACTTGGAGCAGCGTTACGGCAAAAAAACATC
ACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCG
1 14VAADIGAGL AflALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGABKT 51 YGNGIPSLNTG KLKNDKVSRF DFIRQXSVDG QLITLESGEF QVYKQSHSAL 101 TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFD.KLPE GGRATYRGTA 151 FGSDDAGGYL TYTI:DFAAXQ GNGKIEHLKS PEILNVDLAAA DIKPDGRRMA 201 VISGSMLYNQ AnKGSYSIJGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL 251 DGGGGTGSSD LANDSFIRQV LDRQHFEPDG KYHLFGSRGE LAERSGHIGL 301 GKIQSHQLGN LXMIQQAAIKG NIGYIVRFSD HGHEVI4SPFD NH{ASHSDSDE 351 AGSPVDGFSL YRIHWDGYEH HPAflGYDGPQ GGGYPAPKGA RDIYSYDIKG 401 VAQNIRLNLT DNRSTGQR.A, ORFHNAGSM~L 'rQGVGDGFKR ArRYSPELDR 451 SGNAAEAFNG TA~flVKNIIG AAGEIVGAGD AVQGISEGSN IAVMHOGLGLL 501 STENKMARIN DLADMAQLKD YAAAAIRDWA VQNPNAAQGI EAVSNIFMAA 551 IPIKGIGAVR GKYGLGGITA HPIKRSQ4GA IALPKGKSAV SDNFADAAYA 601 KYPSPYHSRN IRSNLEQRYG KENITSSTVP PSNGYJAVKLA. DQRHPKTGVP 651 FDGKGFPNFE ICHVKYDTLEH HHHHH* Example 5 hybrids of 287 Expression of 287 as full-length with a C-terminal H1is-tag, or without its leader peptide but with a C-terminal His-tag, gives fairly low expression levels. Better expression is achieved using a N-terminal GST-fusion. As an alternative to using GST as an N-terminal fusion partner, 287 was placed at the C-terminus of protein 919 ('919-287'), of protein 953 ('953- 287'), and of proteins 0RF46.1 ('0RF46.1-287'). In both cases, the leader peptides were deleted, and the hybrids were direct in-frame fusions.
To generate the 95 3-287 hybrid, the leader peptides of the two proteins were omitted by designing the forward primer downstream from the leader of each sequence; the stop codon sequence was omitted in the 953 reverse primer but included in the 287 reverse primer. For the 953 gene, the 5' and the 3' primers used for amplification included a NdeI and a BamHI restriction sites respectively, whereas for the amplification of the 287 gene the 5' and the 3' primers included a BamHI and a XhoI restriction sites respectively. In this way a sequential directional cloning of the two genes in pET2ib+, using NdeI-BamlH (to clone the first gene) and subsequently BamnH-XhoI (to clone the second gene) could be achieved.
WO 01/64920 PCT/IB01/00420 -23- The 919-287 hybrid was obtained by cloning the sequence coding for the mature portion of 287 into the XhoI site at the 3'-end of the 919-His clone in pET21b+. The primers used for amplification of the 287 gene were designed for introducing a Sal restriction site at the and a XhoI site at the of the PCR fragment. Since the cohesive ends produced by the Sall and Xhol restriction enzymes are compatible, the 287 PCR product digested with SaI-XhoI could be inserted in the pET21b-919 clone cleaved with Xhol.
The ORF46.1-287 hybrid was obtained similarly.
The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens: Mixture with 287 Hybrid with 287 919 32000 16000 953 8192 8192 ORF46.1 128 8192 Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained for 919-287 and 953-287: 919 953 ORF46.1.
Strain Mixture Hybrid Mixture Hybrid Mixture Hybrid MC58 512 1024 512 1024 1024 NGH38 1024 2048 2048 4096 4096 BZ232 512 128 1024 16 MenA (F6124) 512 2048 2048 32 1024 MenC (Cll) >2048 n.d. >2048 n.d. n.d.
MenC (BZ133) >4096 >8192 >4096 <16 2048 Hybrids of ORF46.1 and 919 were also constructed. Best results (four-fold higher titre) were achieved with 919 at the N-terminus.
Hybrids 919-519His, ORF97-225His and 225-ORF97His were also tested. These gave moderate ELISA fitres and bactericidal antibody responses.
As hybrids of two proteins A B may be either NH 2 -A-B-COOH or NH 2
-B-A-COOH,
the "reverse" hybrids with 287 at the N-terminus were also made, but using AG287. A panel of strains was used, including homologous strain 2996. FCA was used as adjuvant: WO 01/64920 PCT/IB01/00420 287 919 287 953 287 ORF46.1 Strain AG287-919 919-287 AG287-953 953-287 AG287-461 46.1-287 2996 128000 16000 65536 8192 16384 8192 BZ232 256 128 128 <4 <4 <4 1000 2048 <4 <4 <4 <4 <4 MC58 8192 1024 16384 1024 512 128 NGH38 32000 2048 >2048 4096 16384 4096 394/98 4096 32 256 128 128 16 MenA (F6124) 32000 2048 >2048 32 8192 1024 MenC (BZ133) 64000 >8192 >8192 <16 8192 2048 Better bactericidal titres are generally seen with 287 at the N-terminus.
When fused to protein 961 [NH 2 -AG287-961-COOH sequence shown above], the resulting protein is insoluble and must be denatured and renatured for purification. Following renaturation, around 50% of the protein was found to remain insoluble. The soluble and insoluble proteins were compared, and much better bactericidal titres were obtained with the soluble protein (FCA as adjuvant): 2996 BZ232 MC58. NGH38 F6124 BZ133 Soluble 65536 128 4096 >2048 >2048 4096 Insoluble 8192 <4 <4 16 n.d. n.d.
Titres with the insoluble form were, however, improved by using alum adjuvant instead: Insoluble 32768 128 4096 >2048 >2048 2048 961c was also used in hybrid proteins (see above). As 961 and its domain variants direct efficient expression, they are ideally suited as the N-terminal portion of a hybrid protein.
Example 23 -further hybrids Further hybrid proteins of the invention are shown in the drawings and have the sequences set out below. These are advantageous when compared to the individual proteins: OR4A6.1-741
ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCAC
CTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGC
AACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTC
CATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTAC
CGCATCCATTGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCC
AAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGC
AGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAA
CGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTT
AAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATT
WO 01/64920 PCT/IBOI/00420
GCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAA
CTCAAGACTATGCCGAGCAGCCATCCCGATGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGA&GCCGTC
AGATTTTTGACACCACAGGTGGGTTCGGAAAGCTGCGACC
GCCTCACACGCCGTGCCACCTTCGAGGATCCGCGGCATTC
GAGGCTCCAAACGCCTACTCCGATTCTCACTGGACTAGCA
GuAAAACATCACCTCCT'CAACCGTGCCGCCGTCAAACGGAAAAATGTCAAACTGGCAGACCAACGCCACCCAAGACA
GGCGTACCGTTTGACGGTAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGGGGTGGT
GTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAGGTTTGCAGTCT
TTAGTGTATCTAGAACAAATAGTGGCCAGGGAAAATAGAA
GGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAGTGGAC
GGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACC
GAGCAATACAAGATTCGGAGCATTICCGGGAAGATGGTTGCGACQCCAGTTCAGAATCGCGACATAGCGGGCGAA
CATACATC LTTTGACAAGCTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGC
GGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGACGGCAAATCGACATTTGATCGCCAGAACTC
AATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCAGCCGTCATCAGCGGTTCCGTCCTTTACAAC
CAAGCCGAGAAAGGCAGTTACTCCCT'CGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAA
ACCGTAAACGGCATACGCCATATCGGCCTTCCGCCAGCAACTJCGAGCACCACCAj2CACCACCACTGA 1 MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ 51 LGNLMIQQAA IRGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG 101 PSLYRIHWDG YEHHPAflGYD GPQGGGYPAP KGARIXYSYD IKGVAQNIRL 151 NLTDNRSTGQ RLAflRPHUAG SMdLTQGVGDG FKRATRYSPE LDRSGNAAEP.
201 PNGTAflIVKN IIGAAGEIVG AGDAVQGISE GSNIAVM2HGL. GLLSTENKMA 251 RINflLAfMAQ LKDYAAAAIR DWAVQZ4PNAA QGIEAVSNIF l4AAIPIKGIG 301 AVRGKYGLGG ITAHPIKRSQ 14GAIALPKGK SAVSDNPADA AYAIKYPSPYH 351 SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KJAflQRHPKT GVPFDGKGFP 401 NEKEVICYDT GSGGGGVAAfl IGAGLAflALTAPLDHKDKGL QSLTLDQSVR 451 IKNEKLKLAAQGA.EKTYGNGD SLNTGKLKND KVSRPDFIRQ IEVDGQLITL 501 ESGEFQVYKQ SHiSALTAPQT EQIQDSEHSG KMVAICRQFRI GDIAGEHTSF 551 DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV 601 DLAAAflIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK 651 TVNGIRRIGL AM(QLEHRHH HH* ORFA6.1-961 ATGTCAGATTTGGCAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGr.AAATACCAC.
CTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGC
AACGTATACGCGCTAAGATTGGTCTGCGTTCGTAGGAGAT
CATCCCCCTCGACACCATGCCTACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGTTTAGCCTTAC
CGCATCCATTGGGACGGATACGACACCATCCCGCCGACGGCATGACGGCACGCGGCGCTATCCCGCTCCC
AAAC-GCGCGAGGGATATATACACTACACATAGCGTTGCCCAAATATCCGCCTCACCTGACCGACACCGC
AGCACCGACAACGGCTGCCGACCGTTTCCACATGCCGGTAGTATGCTGACGCAGGAGTAGCGACGGATTCAAA
CGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTT
AAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGATAjGCGGGCTCAAACATT
GCTGTCATGCACGGCTTGGTCTGCTTTCCACCGAAACAAGATGGCGCGCATAACGATTTGGCAGATATGGCGCAA
CTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAACCCCAATGCCGCACAAGGCATAGAAGCCGTC
AGCAATATCTTTATGGCAGCCATCCCCATCAAGGGATTGGAGCTGTCGGGGATACGGCTGGGCGGATCACG
GCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAALTTTTGCC
GATGCGGCATACG CCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGcAGcG.TTAGGCAAA
GAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACCCACCCGAAGACA
GGCGTACCGTTTGACGGTAAGGGTCCGAATTTTGAGAAGCACGTGAATATGATACGGGATCCGGAGGAGGAGGA
GCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTrGCTGCTGCCTACAACAATGGCCAAGAAATCAAC GGTTTCAAAGCTGr.AGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAXAAAAGACGMACTGCAGCCGAT
GT]YGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAA
CAACTGTCAATAGTCGACGATAAAGTAACAGTG~.CCGTC
GCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTT
GCTGAAGAGACTAAGACAAATATCGTAAAATTGATGAAAATTAGAGCCGTGGCGATACCGTCGACAAGCATGCC
GAAGCATTCAACGATATCGCCGATTCATTGGATGAACCAACCTAAGGCAGACAACCGTCUAACcrGcCAATGAA
GCCAAACAGACGGCCGAAGAACCAACAAACGTCGATGCCAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCC
GAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCGTTACCGACATC AAA3CT GATATCGCTACGAACAAAGATAATATTGCTAAGCAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGc AAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGA3AATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATT
GCCGATCACGATACTCGCCTGCGGTTTGGATZACATGTCAGACCTGCGCAAAAAACCCGCCAAGGCICTTGOCA
GACACGGTTCGCGTCACTCAGTGTGTCAGACGTCGCGGCA
AAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTC
GGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCAC,
TGA
WO 01/64920 PCTIBO1IOO420 -26- 1 MSIDLAXDSPI RQVLDRQHFE PDGKYHLFGS RGELAEIRSGH IGLGKIQSHQ 51 LGNLMIQQAA IKGNIGYIVR FSDHGHBVHS PF'DNHASESD SDEAGSPVDG 101 FSLYRIHWDG YEHEPADGYD GPQGGGYPAP KGARflIYSYD IKGVAQNIRL 151 NLTD1NRSTGQ RLADRFHiAG SI4LTQGVGDG FKRATRYSPE LDRSGNA2hBA 201 FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENIM 251 InMLAUMQ IacDYAAAAXR DWAVQNPNAA QGIEAVSNIF MAAXIPIKGIG 301 AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNPADA AYAICYPSPYH 351 SRNIERSNLEQ RYGXENIETSS TVPPSNGIWV KLAflQRHP1T GVPPDG.I(GFP 401 NFEKHVKYDT GSGGGGATNfl DDV3CKAATVA IAAAYN'NGQE INGFRAGETI 451 YDIDEDGTIT KKDATAAflVE ADDFKGL#GLK KVVTNLTKTV NENKQNVDAI( 501 VYMAAESEIEK LTTKLADTDA ALAflTDAALD ATTNALNKLG ENITTFAEET 551 KTNIVKIDEK LEAVADTVDR HARAFNDIAD SLDETNTKAfl EAVKTPANEAK 601 QTAflETKQNV DAI(VKAAETA AGKAEAAAGT ANTAAflKAEA VAMCVTDIKA 651 DIATN.KDNIA KKANSADVYT 1REESDSKPVIR IDGLNATTRK LDTRLASAEK 701 SIADHflTRLN GLDKTVSDLR KBTRQGLABQ AALSGLFQPY NVGRFNVTAA 751 VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY HVGVNYEWJLE 801 aHHHi* 0RP46.1-961c
ATTAATGCACATTTACGCGTCTGCGCGATCACCAGGATCA
CTTCGACGGGACTCGGGACGCAACGTGGAATCAGCTATGG
AACGTATACGCGCTAAGATTGGTCTGCGTTCGTAGGAGAT
CATCCTCAACAGCCCTCGTCGAGACGTGCCTGCGTTGCTA
CGACATGAGAAGAACTCGCAGGTTAGGCCGGGCGTTCGTC
AAGCCAGAAAAACAGCTAAGGTGCAATTCCTACTACAACG
AGACGCAGCTCGCGTCAATCGGATTCGCCAGGAGGCGTCA
CGGCCCAAACCGGTGCGTGGATCOCAGCTACGATCGTTrT
AAACTACGGGCGAAATTGCCGGGTCGGAGCTACAGCCACT
GCGCTCCGTGGCGTTCCGAAAAAGCCCTACATGCGTTGGA
CTAAATTCOACGCTCCATGCGCAACCATCGAAGCTWGCT
AGATTTTTGACACCACAGGTGGGTTCGGAAAGCTGCGACC
GCCTCACACGCCGTGCCACCTTCGAGGATCCGCGGCATTC
GATGCGGCATACGCCAATACCCGTCCCCTTAC
CATCCCGATTCTCAATGACGCTAGTA
GAACTACCTACGGCCGCACGAAAGC7ATGAACAGCCCAGC
GGGACTTAGTAGGTCCATTGGACCTAAAGTCGACGAGGAG
GCAA-CAGCAGTAAACGCCGGCATCGTCTCAATGCAAACA
GGTCAGTGGGCACAGCTGTAGAGCCATCAAAGCCATCGCA
GTGACGCATTAGTTGTTAAAGTGGCACTACAACTATAACA
CAACTGTCAATAACGAATTAATGAATACACATACGCCGTC
GCTACGTCGTCGTTGTCACCACCTGAAATG~,AAAACAAT
GCGAAATAAAAACTAATGTAAATAAGCTGTAACTGCACTC
GAGAMAGTTGCATATGT- CAAATAGAACMCrTAACGCAGA
GCAAAAGCGAAACACAAGCAGCAATAACGAAATCGAGAAC
GAGTCGTGAACATCGACGCAGCGACGCCGAAGTCGCTAAC
GAACCAGAAAAATTGTAAACAAATCGCTTCCAAAGGCGCG
AATTTAATGTGCGAGTCACAAATGCCCCTGTCGTAA7TCT
GCGTAGTCCCTACGTGAAACGTTAACGGAAAACGCAGCTC
GACACGGTTCGCGTCACTCACTGTTGGACCACCACCG
1 MSDLANDSPI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ 51 LGNLMIQQAA IRGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG 101 FSLYRIHWDG YEHiHPADGYD GPQGGGYPAP KGARflIYSY2 IXGVAQNIRL 151 NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA 201 FNGTAflIVKN IIGAAGEIVG AGDAVQGISE GSHIAVMHGL GLLSTENKMA 251 RINDLAflMAQ LKflYAAAAIR DWAVQNPNAA QGIEAVSNXF MAAIPIKGIG 301 AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFAflA AYAKYPSPYH 351 SRrNRSNLEQ RYGKEN'ITSS TVPPSNGKiqV KLAflQRHPCT GVPFDGKGFP 401 NFEKMVKYDT GSGGGGATND DDVKI(AATVA IAAALYNNGQE INGFKAGETI 451 YDIDEDGTIT KKDATAADVE AfDPKGLGLK KVVTNLTKTV NENKQNVDAI( 501 XflAAESEIEK LTTKLAflTDA ALADTDAALD ATTNALNKLG ENIT'rFAEE'r 551 KTNIVKIDEK LBAVADTVDK HAEAFNDIAD. SLDETNTKAfl EAVXTANBAX 601 QTABETKQNV DAKVKAAETA AGKAEAAAGT AN'TAADKEA VAMKTDIKA 651 DIATNKDNIA KRPJSAflVYT REESDSKFVp. IDGLWNATTEK LTLASAEK 701 SIMfHDTRLN GLDKWVSDLR KETRQGLABQ AALSGLFQPY NVGLEHHHH 751 H* WO 01/64920 PCT/IBOI/00420 -27- 961-0RF4 6.3.
ATGCCACAGCAGTAAACGCCGTGCTGTCGCAACAGCAGAT
AAGTTAACGAAACTTCAATAGAGCGAATACAAAAGACGAC
GAGTAGCAGCTAAGCGGCGAAAGCTATACGCAACGCAGAA
AACAAGCAGCAGAAGTCGACGAAAAAGTAACAGTGAAATA
GCGTTGAAATAGCCC~.TCACACAGCTATATGAAATTAGC
TTGTAGGCAGCATT
GGCGTCGCAAGA
GCGACTCAGTTGCATATGTAACAATAGCrAGACGCAACCAT
GAGCACGCGCAGACAAAACTGAGCAGAAGTCGACGACGCA
GCGACGCCGCCGTAATCGCAAAGCAGTTGTCAATACAACA
GCGTTGTCACAGTAATCAAAGCACGGCAGGAACGGAATTA
AGAATGCGATAGTTACCATCGAAATGCCCCTGTCGTAAAC
ATGCACCAATGCGAGTTGTAACGGCGCTCCAGACCCAGCT
GCG-CLGCCCCCGTTTCACTAACTGTCMALGACG~.ATGCG
TAAACGACG~.CCACGACGTCGTTCGAATTCGCAGAGGGC
GTGCCTGCGTCTCCGCACTTGCTATAGGGGACG~.AGGAC
GATTGAAGTCTTTCGAGTTG~c.CGATCACCAGGATCACAT
GGACGGGACTCGGGACGCTTGGTGGAATCAGCTATGGACT
ATATACGCGCTAAGATTGCAATGCGTTCGTAGGAGATCTC
CCTCAACAGCCCTCGTCGT3ACGTGCCTGCGTTGCT~CCT
CATGAGAAGAACTCGCAGCAGAGGCCGGGCGTTCGTCAAG
GCAGAAAAACAGCTAAGGaGCAATTCCTACTACAACGACC
GGCAGCTCGCGTCAATCGTGATCGCCAGGAGGCGTCACCC
ACCAAACCGGTGCGTGGATCGCGACTCAGCCGAAACTAAA
ATACGGGCGAAATTGCCGCAGCTCCGCTACAGCCACTGTT
ATCCGTGGCGTTCCGAAAGTGrCCTACATGAAACGGACCA
GATTCCCG~.CTCCATGCGCAAACCAGCCCAGAAAGCTACA
ATTTTGACACCACAGGTGACGTCGGAAAGCTGCGACCGAA
CCACACGCCGTGCCACCTGCAA~GATCCGCGGCATTCGTC
GCTCCAAACGCCTACTCCA6TTCTCACTGGACTAGCAGAA
ATACCTACGGCCGCACGAAAGTAATGAACACCACGAAACT
CCTTACrTACGTTCATTGGAACTAAAGTCCCACCACCACCA
TGA
1 MATNDDDVKUK AATVAIAAAY NNGQEINGPX AGETIYDI13E DGTITKCDAT 51 AADVEADDFKC GLGLKKVVTN LTKTVNENKQ NVDAXVKAAE SEIEKLTTKL 101 ADTDAALAflT DA?.LDATTNA LNKLGENITT FAEETKTNIV KIDERLEAVA 151 DTVDKHAZMF NDIADSLDET NTKADEAVKT AMEQTAEE TKQNVDAKVK 201 AAETAAGKAE AAAGTANTAA DKAEAVAARV TDIKADIATN KDNIAXKMANS 251 ADVYTRIEESD SKFVRIDGLN ATTEKLDTRL ASAEKSIAflH DTRLNGLDKT 301 VSDLRIKETRQ GLAEQAALSG LFQPYINVGRF NVTAAVGGYK SESAVAIGTG 351 FRFTENFAAI( AGVAVGTSSG SSAAYHVGVN YEWGSGGGGS DLANDSFIRQ 401 VLDRQHFEPD GKYHLFGSRG EL-AERSOHIG LGKIQSHQLG NILMIQQAAXK 451 GNIGYIVRPS DHGHEVHSPF DNHASHSDSD E.AGSPVDGPS LYRIHWDGYE 501 HHPAflGYDGP QGGGYPAPKG ARDIYSYDIK GVAQNIRLNL TDNRSTGQRL 551 ADRFHil'AGSM LTQGVGDGPF( RATRYSPELD RSGNAAEAPN GTADIVKNII 601 GAAGEIVGAG DAVOGISEGS NIAVMHSGLGL LSTENKKARI NDLADMAQLYK 651 DYAAAAIRDW AVQNPNAAQG IBAVSNIFM4A AIPXKGIGAV RGKYGLGGIT 701 AHPIKRSQXG AITALPKGKSA VSDNFADAAY AXYPSPYHSR NIRSNLEQRY 751 GKENITSSTV PPSNGKNVKTJ ADQRHPKTGV PFDGKGFPNP EKHVKYDTLE 801 jHHjfl* 961-741
ATGCCAAGCAGTTAAAGTCATTGCTGTCGCAACAGCAGAT
AAGTTAACGAAACTTCAATAGAGCGAATACAAAAGACGAC
GAGTAGCAGCTAAGCGGCGAAAGCTATACGCAACGCAGAA
AACAAGCAGCAGAAGTCGACGAAAAAGTAACAGTGAAATA
GCGTTGAAATAGCCCGAGACACACCTGAAATGAAATTAGC
TTGTAGGCAGCATTGAAATAGAAATGACGGCGTCGCAAGA
GCGACTCAGTTGCATATGTAACACCAGCGCAGCTAACGCA
GAGCACGCGCAAACACAAGCAGCAGAAGTCGACGACGCA
GCGACGCCGCCGTAATCGCAAGCGACGCCGAAGTCGCTAL
GCGTTGTCACAGTAATCAAAGCACGGCAGGAACGGAATTA
AGAATGCGATAGTTACCTCGTAAAC
ATCCGTAGTCCCTACGTGAAACGGCGCTCCAGACCCAGCT
WO 01/64920 PCT/IBOI/00420 -28-
GCGAAGCCCCCGTTTCACTAAAGGOCGTATTAGCGATGCG
TAAACGACGATGCTGTCGCTCGTTCGAATTCGCAGAGGGC
GTGCCTGCGTCTCCGCACTTGGGCATCATGGTCGGGGGTT
GCGCAACGGGGCTCGTCCACCCCGTGCAAAAAGTGATTT
ACC~-TATCTAGAACAAATIAGTGGCCAGGGAAACTTGACG
GAACTATCGCATGAACAAGTAGCTTGCTACGCATGATGCG
CACCTACTGGGGAATCAGAAAACAGCTCGCTACCTTAACA
CA-TCAATGACTCGGAAGTGGACCATCGACGGCTGGGGAA
ACTTTGCACTCGAGGCGGGCT.CCGAGCTCGTAAGTCGCG
AACGCTCCAAATCCGCACGGAAGCAACACTTAACCAACCA
GTGCTGCCGCAACACGAGAACGCTCGCTACGTCTCTAACA
GCGGAGCGTCCCCGACTGCGAAGCAGATGCGACCGATAAC
GTACGAAGCTTGCTG~.CACATGGACCACCACCG
1 MATM~DDVXK AATVAIAAAY NNGQEINGFIF AGETIYDIDE DGTITKKDAT 51 AADVEADDYK GLGLKKWTN LTKTVNENKQ NVDAXVKAAE SEIEKLTTKL 101. AElTDAALAflT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA 151 DTVDRHARAF NDIADSLDRT NTKADEAVKT ANEAQTABE TKQNVDAXVK 201 AAETAAGKM AAAGTAXTAA DKAEAVAARV TDIKADIATN KDNIAKKANS 251 ADVYTREESD SKFVRIDGLN ATTEXLDTRL AS?.EKSIAflH DTRLNGLDKT 301 VSDLRRETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG 351 PRFTENFAAI( AGVAVGTSSG SSAAYHVGVN YEWQSGGGGV AADIGAGLAD 401 ALTAPLDHKD KGLQSLTLDQ SVRKNEKLKL AAQGAEKTYG NGDSLNTGKL 451 KNDKVSRFDF IRQIEVDGQL ITLESGErQV YKQSHSALjTA FQTEQIQDSE 501 HSGKMVAICRQ FRIGDIAGEH TSFDKLPEGG RATYRGTAFG SDDAGGKLTY 551 TIDFAAXQGN GK(IEHIJISPE LNVDLAAADI KPDGKRHAVI SGSVLYNQAE 601 KGSYSLGIFG GKAQBVAGSA EVKTVNGIRHi IGLAA KQLEH HHHHH* 961-983
ATGCCACAGCAGTAAACGCCGTGCTGTCGCAACAGCAGAT
AAGTTAACGAAACTTCAATAGAGCGAATACAAAAGACGAC
GArTGACGCATTAGTTGTTAAAGCTATACGCAACGCAGAA
AACMCTGTCAATAACGAATTAAAAAGTAACAGTGAAATA
GCGTTGAAATATCGTTGTCACCC
GCTATATGGAGAAATATAACGACA
TTGTAGGCAGCATTGAAATAGAAATGACGGCGTCGCAAGA
GCGACTCAGTTGCATA~,AGACACCAGCGCAGCTAACGCA
GAGCACGCGCAGACAAAACTGTCAATAAC~-GACGACGCA
GCGACGCCGCCGTAATCGCAAAGCAGTTGTCAATACAACA
GCGTTGTCACAGTAATCAAAGCACGGCAGGAACGGAATTA
AGAATGCGATAGTTACCATCGAAATGCCCCTGTCGTAAAC
ATTGCCGATCACGATACTCGCCTGACGGTTTGGATCAGTGTCAGACCTGCGGCCCGCCAAGGCCTT
GCGAAGCCCCCGTTTCACTAAAGGGCGTATTAGCGATGCG
TAAACGACWGCCACGACGTCGTTCGAATTCGCAGAGGGC
GTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGTCTTACGAGGATCCGAGGGACT
TCGGCGCTATCGCGACGACGAGACGAACAACGGATACGAT
TCTCCGTTAGAGATTCALAAAGAGTTTCGTGGTAGTCGTC
GAAGAGCAACArCCCCCGACGCTCGAATTCACCA~.CCTCA
AATGTACTAACGATGACGCAACGAGGGTGGTGTTGCAAAG
GATCTGCGAACTTCGATTTGAAKGAAGCAACAATAAAACA
ACGGAAGGAGAGGCGAAGAGrGAAAATAGTCTCAGTAGCT
ATGGCGACAGCAGAACGCCTAAGATGAAACATGTTCAATT
GGGGGTCTGCGAACGAGGTTGCCCAGGCCAAAATATCATA
GAACAACAAGTGTCGCTCCAGCTGTAGTGCACTGGGGACT
AATAACAGTTGGAACACATCGAGGGCAGGCACTGCCGACCTTTTCCATAGCCATTCGGAGGAGCGTACCGC
CAGGTCCATTCGCGGTAAAAGAGTTCCTAGACGGGTAGCA
CTTCACCTCTAAAAAGTTCTTTTGCGCAGCCCACCGCACC
TAGCTTGCTTAGAAGCCCAAGGATTAATGAGGAACCGGAA
AATC-CGAAGAGA~CGGAAACCCTATTGTCACTGGATATC
ATTGGCGCGACTTAGAGGCGTTACGAAACGTCATGCGAAC
TTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGATACCCGTGGATGAGCCGACCTGCGT
ACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGACGAAGTTCGGCTGGACTGTGGATGCG
GGAGCAGAGACGGCTTCTCGGATTCGCAAGAGTCTCAATC
TATCTCTAGCTTAGAGGGCTATAAAGCGACACGACGAGCA
AAACAAGGAACATTGAGGTCCTGGTTCOACAAACGTTCCT
GAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGACAGCGCGGCATTGTCTATCTGGCA
r.TCGCATCGGAAGACGAAACAAGATTCGTGcGAArTCCGA WO 01/64920 PCTIBO1I/0420 CCCGAGACGGTTGAACTGCGGCAGCCACCGCACAGATATGCCGGGCATCCGCCCCTACCGCCCCGCr-
GCGACGAACTCATCGCAGTTCGACTACGCCCGTCGCAGCA
AGACCGCAOCAAGAGAGCCtGAACGACG~.GTGCAACGAGG
CTCCTACCCACCAAGCG~.AGTGACGGGTTGAGAAGGGCG
ACCACGCGATCGGAACGGAAACAACGCCAATGCTGAGACC
TGACAACGGAAGAAACAACTAGCGTGAGAAGCCAGGGGTT
GGTTTAAGCGTTCAGAGTCAAAACTACGACCGTCGCACTC
GAAGGCACGTCAACGGCACGCGrATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGGCCrAAGGAGAT TTAGTGAGGTTC3TCACGTAACGAGATGCAAAG~.~TTGcG
AGGCAACTATAGCCCGTGATGCGTTAGTTGACCTACAAAC
GTCGTGACGGGGGACCACGAGGCCATCCGACGCGTTCGCC
ACGACACGAGCGGCCCAAGCCAACGCGTGCGCGGGGAGCA
TTGCAGCGACGTGCCTAACAGCGTCAAATCGACAACGCAT
GGCGTAGGCTACCGTTCCTCGAGCACCACCACCACCACCACGA
1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 HATNDDDVKK AATVAIAAAY I'JGQEINGFK AADVEADDFK GLGLKKVTTN LTKTVNENKQ ADTflAAI.ADT DAALDATTNA LNKLGENITT AGETIYDIDE DGTITKKflAT WJDAKVKAAR SEIEKLTTKL FAEETKTNIV KIDEKLEAVA
DTVDIGHAEAF
AAETAAGYAE
AflVYTREESD
VSDLRKETRQ
FRPFTENFAAX
GIGSNSRATT
PPPNLHTGDP
SFPELYGRKE
AKPTDI.RHVK
FNEMMVAAIR
QALLDYSGGD
NTYALLPFYE
GITAMWCLSA
SNDNLRTTLL
TKGTSDIAYS
.YGNNKSDMRV
GSLQLDGKGT
PFLSAAKIGQ
AARTASAAAH
ADRTDMPGIR
DMQGRRLKAV
GIAAKTGENT
LKGLFSYGRY
EJTVEGGLRYD
KAVLPATAGV
ADVEFGNGWN
NDIADSLDET NTKADBAVKT ANEAKQTAE TKQ~1vDAKvK AAAGTAINTAA DKAEAVAAIKV TDIKADIATN KDNIAKKANS SKFVRIDG*N ATTEKLDTRL ASAEKSIAflH IYIRLNGLDKT GLXAEQAALSG LFQPYNVGRF NWPAAVGGYK SESAVAIGTG AGVAVGTSSG SSAAYHVGVN YEWGSGGGGT SAPDFNAGGT AKSAAVSYAG IKNEMCKDRS IdLCAGRDDVA VTDRDfAKINA PNPNDAYKN.L INIJKPAIEAG YTGRGVEVGI VDTGESVGSI HGYNBNYKNY TAYMURAPE DGGGKDIEAS FDDEAVIRTE EIGHIDLVSH IIGGRSVDGR PAGGIAPDAT LHIMTNDET NAWVKLGERG VRIVNNSFGT TSRAGTADLF QIANSEEQYR KTDEGIRIMQ QSDYGNLSYH IRINKNMdLFIF STGNDAQAQP KDAQKGIITV AGVDRSGEIF KREMYGEPGT EPLEYGSNHC PYEASVRFTR TN1'IQIAGTS *FSAPIVTGTA ALLLQKYPWM TTAQDIGAVG VDSKPGWGLL DAGKAMNGPA SFPFGDFTAD FRI'WISGTGG LIKKGGSQLQ LHGNNTYTGK TIIEGGSLVL ETKGALIYNG AASGGSLNSD GIVYLADTDQ SGANETVHIK LYTRLGKLLK VDGTAIIGGI( LYMSARGKGA GYLNSTGRRV DYSFFTNIET DGGLLASLDS VEKTAGSEGD TLSYYVRRGN .SAPAGLK{AV EQGGSNLENL MVELDASESS ATPETVETAA PYGATFRAAA AVQHANAADG VRIFNSLAAT VYADSTAAI{A SDGLDHNGTG LRVIAQTQQD GGTWEQGGVE.GKMROSTQTV TAAATLGMGR STWSENSANA KTDSISLFAG IRHDAGDIGY KNSISRSTGA DEHAEGSVNG TI.MQLGALGG VNVPFAATGD LLK(QDAPAEK GSALGWSGNS LTEGTLVGLA GLKLSQPLSD ERDLNGRDYT VTGGFTGATA ATGKTGARNM PHTRLVAGLG GLARYSYAGS KQYGNHSGRV GVGYRFLEHH HHHH* 961c-0RP46.12
ATGCCACAGCAGTAAACGCCGTGCTGTCGCAACAGCAGAT
AACrTTAGT~.GGCACAGCTGTAGCGAATACAAAAGACGAC
GAGTAGCAGCTAAGCGGCGAAAGCTATACGCAACGCAGAA
AACAAGCAGCAGAAGTCCATTAAAAAGTAACAGTGAAATA
GCGTTGAAATAGCCCGAGACACACCTGAAATG.GAAAAAGC
TTGTAGGCAGCATTGAAATAGAAATGACGGCGTCGCAAGA
GCGACTCAGTTGCATATGTAAC;CCAGCGCAGCTAACGCA
GAGCACGLGCGAAACACAAGCAGCAGAAGTCGACGACGCA
GCGACGCCGCCGTAATCACGCAGCAGTTGTCAATACAACA
GCGTTGTCACAGTAATCAAMCACGGCAGGAACGGAATTA
AGCAAATTTGTCAGAATTGATGGTCTACGCTACTACCGAATTGGACACACGCTGTTCTGCTGAAAATCC
ATGCACCAA~GCCMCrTTGTXACGGCGCTCCAGACCCAGCT
GCGAAGCCCCCGTTTCACTAAAGGGGACGAGGAGTAATGC
AACGATTCTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGACCCGACGGGAATACCACCATTCGGCAGCAGG
GGGGAACTTGCCGAGCGCAfCGCCATATCGGATTGGAAAATAAAAGCCATCAGTTGGGCAACCTGATATTCAA WO 01/64920 PCTAEB01/00420
CAGGCATAGAAACGTCTTCCTTCACCGCCAGCATCCTCA
AACTCTAAT~.TCGTACGTGTCGTAGATACTTCGACATGA
GGTCGAACTCGCAGCTTAGGCCGGGCGTTCGTCAAGGG~.A
ATTCGTCAAAAGCTGCAATTCCTA.CTA.GCACCGACGCAG
CTGCACTTCCAGCGATTCGCCAGATGCAGATAAGGCCCAA
AGCCACGAAACGCAGCCGACTCAGCCGAAACTAAAAcTrG
GCGAGGATGCGGAGGTCGGAGGAAGGAGTAAATCGCTCCG
TTGTTCTCACAACAAGCCCTAAGTTGAAAGCCATAAATTC
CACGCTCCATGCGCAACCATGCCCAGAAAACGCGATTTTT
GCGCTCCTAAGATGGTTCGGAATCGTGGGCTAGCCTCACA
1 MAAGGTTNDDVKAATAAAAY bGAAQEINGK ACGETCGAGCACGTITKATCACCG 51 AAflVEAXlDFKX GLGLKKVVTN LTKTVNENKQ NVDAKVY(AAE SEIEKLTTKL 101 AflTDAALADT DAALDATTNA LNKIGENITT FAEE.TKTNIV KIDERKjEAVA 151 DTVflKHAEAp NDIADSLDET NTKADEAVI(T AkMEAQTAEE TKQNAKXcv 201 AAETAAGKAE AAAGTANTAA IDYAEAVAAKV TDIKAflIATN KDNIAKKANS 251 ADVYTRBESD SKFVRIDGLN ATTEKLDTRL ASAEKSIAflH DTRLNGLD)KT 301 VSDTJHIETRQGILAEQAALSG LFQPYNVGGS.GGGGSDLAN
SFIRQVLRQ
351 HFEPDGKYHL FGSRGEIJABR SGHIGLGKIQ SHQLGNLMIQ QAAIXGNIGY 401 IVRFSDHGHE VHSPFDNHAS HSDSDEAGSP VDGFSLYRXH WDGYEHHPAD 451 GYDGPQGGGY PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH 501 NAGSMLTQGV GDGPXRATRY SPELDRSGNA AEAFNGTADI VXNIIGAAGE 551 IVGAGDAVQG ISEGSNIAVM. HGLGLLSTEN IQMARINDLAfl MAQLKDYAAA 601 AIRDWAVQNP NAAQGIEAVS NIFNAAIPIK GIGAVRGKYG LGGITAHPIK 651 RSQMGAIALP KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI 701 TSSTVPPSNG KNVKJAQRHi PKTGVPFDGK GFPNFEKHVK YDTLHmH 751 H* 961c-741
ATGCCACAGCAGTAAACGCCGTGCTGTCGCAACAGCAGAT
AAGTTAACGAAACTTCAATAGAAGCCATCAA;AAGACGAC
GAGTAGCAGCTAAGCGGCGAAAGCTATACGCAACGCAGAA
AACAAGCAGCAGAAGTCGACGAAAAAGTAACAGTGAAATA
r.~.TTCCGTCGTCGTTGTCACCACCTGAAATGAAATTAGC TTGTAGGCACCATTGAAATAGAAATrArCGGCGTCGCAAGA
GCGACTCAGTTGCATATGTAACACCAGCGCAGCTAACGCA
GAGCA-AAGCGAAACACALCTCAGCAGAAGTCGACGACGCA
GCGACrCGTGAACATCGACGCAGCAGTTGTCAATACAACA
GCGTTGTCACAGTAATCAAAGCACGGCAGGAACGGAATTA
ACAATGCGATAGTTACCATCGAAATGCCCCTGTCGTAAAC
ATGCACCAATGCGAGTTGTAACGGCGCTCCAGACCCAGCT
GCGAAGCCCCCGTTTCACTAAAGGGGACGAGGTGGCCGCA
ATGTCGGTGCAGATACCCGTGACTAGCAGTTCGCTGCCGA
CATCTAGAACAAATAGTGGCCAGTCGAAATAGAAGTAACT
AAAGGAATAGAGCA;TACGTCATTTCCAACAGGAGGACCT
ACTGAATGGGTCATTCACAGCATCCTACGCTCGCGGAAAA
GATGACTCGGAAGTGGACCATTAATGCAAACGCACTCTTT
GAAGTCCAGCGAGCAAACCGACGGTGTCGCAGCGGAACGC
TAACTGTTGCCAGAGA~GCAATGAATGATGCGATATTGCT
GCGCCGTTAGCGTGA;CCAGCTACGGTCGCTTCACACGGA
GGATATCTGTTTTGGAAGCAGAGTCGCGGGAGGAACTMW
ATCCAACCTGCCAGAATGGACCACCACCG
1 MATNDDDVXIC AATrVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDB'K GLGLKKVVTN LTKTVNENI(Q NVDAIKVKAAE SEIEKLTTKL 101 ADTDAALAn'r DAALDATTNA LNKLGENITT FAEETKTNIV KIDF.KLEAVA 151 DTVDKHAEAP NDIADlSLDET NTKADFlEAVKT AN'EAXQTAEE TKQNVDAKVK 201 AAETAAGA AAAGTANTAA DKAEAVAAXV TDIKADIATN KDNIAKKANS 251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIhflH DTRLN'GLDKT 301 VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGVAAflIG AGLADALTAP 351 LDHKDKGLQS LTLDQSVRKaq EKLKLAAQGA EKTYGNGDSL .ETGKLKNDKV 401 SRPDFIRQIE VDGQLITLES GEFQVYKQSH SALTAFQTEQ XQDSEHSGKOM WO 01/64920 PCTILBO1/00420 451 VAKRQFRIGD IAGEHTSFDK LPEGGRATYR GTAPGSDDAG GKLTYTIDFA 501 AKQGNGKIEH LKSPELNVDL AAAflIRPDGK RHAVISGSVL YNQI4EKGSYS 551 LGIFGGKAQE VAGSAEVKTV NGIRHIGLAA KQLE1MU~fiMM 961c-983
ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAGAAATC
AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAGACGGCACATACOA;AAGACGC1ACTGCGCC
GAGTAGCAGCTAAGCGGCGAAAGCTATACGCAACGCAGAA
AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAGTTACAACCAAQTTAGCAGACACTGAT
GCGTTGAAATAGCCCGAGACACACCTGAAATGAAATTAGC
TTTGCTGAAGAGACTAAGACAAATATCGATTGATGAAATTAGAAGCCTGCTGATACCGCGACAAGCAT
GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAACCAACACTAGGCAGACGAAGCCGTCAAAACCGCCAAT
GAGCACGCGCAGACAAAACTGTCAATAAC~-GACrUCGCA
GCGACGCCGCCGTAATCGCAAAGCAGTTGTCAATACAACA
GCTGATATCGCTACGAACAAAGATAATATGCTAAAAGCAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC
AGCAATTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAATTGG ACACCTTGGCTTCTGAAATCC
ATGCACCAATGCGAGTTGTAACGGCGCTCCAGACCCAGCT
GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGCGGAGGCGGCACTTCTGCGCCC
GATCAGAGCTCGTTGCGACGAGGACAACAACGACGACTCC
GGACAACAAGGAAAAAGAGTTGGCGCGAGCTGGTAAAAGA
GCCAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAATGACGCATACAALGAATTTGATC
AACCTCAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAJATCCGPC
GGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAGAACACGGCTATAACGAAAJTACAAAAACTTACGGCGTAT
ATGCGGAAGGAGCGCCTGAAGACGGAGGCGGTAAAGACATrGAAGCTTCTTTCrACGATGAGGCCGTTATAGALGACT
GAGAACGCGTTCCAGALAAACGCCTGTTGCCCTTATGGGG
TCCGTGGACGGCAGACCTGCAAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAG
AAGATAGTGACACGATCTGTAGTGCACTGGGCCTGCAACG
TTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTCCAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTG
CTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTAC
CAACGATAACTCTTACTTGCGGATAGAAGTACCAAAAGCT
TTCATTTAAAAGTAAAGATTACGCCGCTGCGATGGAATCA
CGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTG TTACTGCGGTC
CTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCA
CCCATCGTAACCGGCACGGCGGCTCTGCTCTGCAGATACCCGTGGATGAGAGACACCTGCGTACCCGTTG
CTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGAGTCGGCTGGGGACTGCTGGATGCGGGTAAGGCC
ATACGCCCTCTCGTGCATTCGCGTCAAGAACGTTGCA
CT
CGTAACGACATTTCAGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTAT
ACGCAACTACAGCGTGTGGTTAGCAACATGAAGGGCAACA
GGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGACCGACGGCATTGTCTATCGGCGATACCGAC
CATCGGAAGACGAAACAGCGCTCGTGCGAAGAGTTCCCTT
GGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGCACCCGCAAGGGGGCAGGC
TACCAATCGAGCTTCCTCGGGCGCAACGCGATTCTCTAAA
ATCGAAACCGACGGCGGCCTGcTGGcT~ccCTCGACAGCGTCGALAACAGCGGCGGAAGGCGACACr.CTGTcc
TATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGACACGCC
GTAGAACAGGGCGGCAGCAATCTGGAP.AACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACG
GTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCC
GTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACGTACCGCC
GCCCATGCCGATATGCAXGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTC
ATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCALACC
GTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAA
AAATCATCAACGCGATGCGTGCGCTCGAGTCGCAACGTTT
AAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGC
GTCACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAIACGTTCCGTTTGCCGCAACGGGAGA~lrGACGGTC
GAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAAC
AGCCTACTGAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATIAAGCCGTCCTGTTT
GCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCA
ACGCAAGGGAGATTCGAACGC~.TGCGCGCCGTTGATGCA
GGCTGGAACGGCT'rGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGC
TACCGGTTCCTCGAGCACCACCACCACCACCACTGA
1 MTNDDDVKK AATVAIAAAY NNIGQEXNGFK AGETIYDIDE DGTITKKDAT 51 AADVXEADDFK GLGLIG(VVTN LTKTVN'ENKQ NVDAKVKAAE SEIEKTTKL 101 ?.DTDAALADT DAALDATTNA LNKLGHNITT FAEETKTNIV KIDERLEAVA 151 nnioiHnEAP 1DIAflSLDET NTKAflEAVKT ANEAKQTABE TKQNVDAICVK 201 AAETAAGKAE AAAG'rANTAA DKAEAVAAKV TDIKADfIATX KDNIAK(ANS 251 A]DVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIAflH D'DRLNGLD)KT WO 01/64920 PCTIBOIOO420 301 351 401 451 501 551 601 651 701 751 801 85~1 901 951 1001 1051 1101 1151 1201 1251 *1301 1351 VSDLRETRQ GLAEQAALSG LFQPYNVGGS GGGGTSAPDF NAGGTGIGSN SRATT).XSAA VSYAGIKNEM CI(DRSMLCAG RfDUVAVTDP.D AKINAPPPNL HTGDFPNPND AYKNTJINLKP AIEAGYTGRG VEVGIVYDTE SVGSISFPEL YGRKEHGYNE NYIQNYTAYR KFAPEDGGOK DIEASFDDEA VIETEAKPTD IRHVKEIGHI DLVSHIIGGR SVDGRPAGGI APDATLHfl4N TNDETKNEAM VAAIRNAWVK LGERGVRIVN NSFGTTSRAG TADLFQIAN~S EEQYRQALLD YSGGDKTDEG IFRLMQQSDYG NLSYHXRNKN M~LFIFSTGND AQAQPNTYAL LPFYEKDAQK GIITVAGVDR SGEKFKREU( GEPGTEPI.EY GSNHCGITAX WCLSAPYEAS VRFTRTNPIQ IAGTSFSAPI VTGTAALLLQ KYPWMSNrhJL RTTLLTTAQD IGAVGVDSKr GWGLLDAGKA 1ME.GPASFPFG DFTADTKGTS DIAYSPRNDI SGTGGLMXG GSQLQLHGNN TYTGKTIIEG GSLVLYGNNK SDMRVETKGA LIYNGAASGG SLNSDGIVYL ADTDQSGANE TVHIKGSLQL DGKGTLYTRL GKLLKVDGTA IIGGKLY1MSA RGKGAGYLNS TGRIRVPFLSA AKIGQDYSFF T1NIETDGGLL ASLDSVEKTA GSEGDTLSYY VRRGNAAIRTA SAAAHSAPAG LKHAVEQGGS NLENLMVELD ASESSATPET VETAAADRTD MPGIRPYGAT P'RAAAAVQHA IIAAflGVRIFN SLAATVYAflS TAAHADMQGR RLKAVSDGLD HNGTGLRVIA QTQQDGGTWE QGGVEGYKMRG STQTVGIAAY( TGEN'DTAAAT LGMGRSTWSE NSANAKTDSI SLFAGIRHDA GDIGYLKGLF SYGRYXNSIS RSTGADEHAE GSVNGTI.MQL GALGGVNVPF AATGDLTVEG GLRYDLLKQD AFAEKGSALG WSGNSLTEGT LVGLAGLKLS QPLSDKAVLF ATAGVERflLN GRDYTVTGGF TGATAATGXT GARNMPHTRL VAGLGADVEF GNGWNGLARY SYAGSKQYGN HSGRVGVGYR FLEMHHHH* 96lcL-ORF46.1
ATAAATTCTCAGATACCGCTCTGCCTCGACGGATGACAAA
GAGCAGTAAAGTCATTGCTGTCGCAACAGCAGATACGTCA
GCGAAACTTCAATAGAAGCCATACAAAAGACGACGTTGAC
GAGCTAAGCGGCGAAATGGCAACGCAACGCAGAAAAAACT
GAGCMTAACGAATTAAAAAGTACACATACGCCGTCGTTGA
GAATAGCCCGAGACACAG~rGATATGGGAAAAC~-TTCGAA
ACAGCATTGAALTGTAAATGACGGCGTCGCAAGAGCAGAT
AAOTTGCATATGTAACAATAGCGCAGCTAAACCATIACAAA
ACrCGAX CAAA.AGCAGCAGAAGTCGACGACGCAGCAGTC
GCGCCGTAATCGCAAGCGACGTGTCAATACAACAGTAACC
ACALAAAATTGTAAACAATCCAGGAACGGAATTAACATTT
AGATAGTTACCATCGAATGAACCCTGTCGTAAACATCGTA
GAATGCGAGTTGTAAATTAACGGAAAACGCAWTGAACAC
GCCCCGTTTCACTAACTGT4TCGAGGAGTAATGCACATTT
ATCG~GTTGCGCGATCACCAGGAAACCTTCGACGGGACTC
GACCG~.CTTGATGAAAAAACACGTGCACGTATACGCGCT
AA~-ATTGCAATTCCTTCACCGCCAGCATCCTCAACAGCC
CATCATTATAGCGATCGTAGATACTTCGACATGAGAAGAA
CACCCGCGTTAGGCCGGGCGTACCCCCAGCCAGAAAAACA
GAAAAGCTGCAATTCCTACTACAACGA;ACGCAGCTCGCG
TTCCAGCGATTCGCCAGGAGGAGATA~GGCCCAAACCGGT
GAAACGCAGCCG~CTCAGCCGCGTTGTAACTACGGrGAGrA
ATGCGCCGCAGCTCGGAAGGAGTAAATCGCTCCGTGGCGT
TCACAACAAGCCCTACATGCGAAGCCATAAATTCGACGCT
CGGTGGATCAACCAGCCCAGATGACGCGATTTTTGACACC
ATAAGATGGTTCGGAAAGCTGGGCTAGCCTCACACGCCGT
GGGGTGATCGAGGATCCGCGGAATTGCAGGCTCCAAACGC
CCTCATCGATTCTCACTGGACOTCGAAAACTACCTACGGC
CCATTGGACCTAATTAAGACCA
1 51 101 151 201 251 301 351 401 451 501 MKHFPSKVLT TAII.ATPCSG AI.AATNDDDV KAATVAIAA AYNNGQEING F7AGETIYDI DEDGTITKKD ATAADVEAflD FKGLGLKKVV TNLTK1'VNEN KQNVDAKVKA AESEIEKLTT IU.AMlDAALA DTDAALDATT NALNKLGENI TTFARETKTN IVRIDERLEA VAflTVDKHAE AFNDIADSLD ETNTKAflEAV KTANEAKQTA EETXQNVDAK VXALAETAAGK AEAAAGTAWT AADKAEAVAA KVTDIKADIA TNKDNIAKKA NSADVYTREE *SDSKFVRIDG LNATTEKLDT 1RLASAEKSIA DHDTRLNGLD KTVSDIJRKET RQGLAEQAAL SGLFQPYNVG GSGGGGSDLA NDSFIRVJD RQHFEPDGKY HL1'GSRGELA ERSGHIGLGK XrQSHQLGM XQQAAIXGNI GYIVR{FSDHG BBVHSPPDNH ASHSDSDRAG SPVDGFSLYR IHWDGYEHHP ADGYDGPQGG GYPAPKGARD IYSYDIKGVA QNXRLNLTDN RSTGQRLADR FHNqAGSMLTQ GVGDGFKRAT RYSPELDRSG WO 01/64920 PCTTB01/00420 -33-.
551 NAAEAFNGTA DIVKNIIGAA GEIVGAGDAV QGISEGSNIA VMHGLGLLST 601 ENIKIARIDL ADMAQLIOJYA AAAIRlWAVQ NPNAAQGIEA VSNIPMAAZIP 651 IKGIGAVRGK YGLGGITAHP IK1RSQMGAIA LPKGKSAVSD NFZADAAYAKY 701 PSPYHSIRNIR SNLEQRYGKcE NITSSTVPPS NGKQNVKLADQ RHPI(TGVPFD 751 GKGFPNEKH VECYDT* 96lcL-741
ATMCCTCACAATCGCAACACTTCATTTTGGCCCGCGCAAA
GAGCAGTAAACGCCGGCATCGCGCAACAGCAGATACGTCA
GCG~-GCACArAATAGAAGCCATACAAAAGACGACGTTGAC
GAGCTAAGCGGCGAAATGGCACTACAACTATAACALAACT
GAGCAGAAGTAATTAAAAAGTAACAT~,AAATAGCCTAC
GAATAGC-TTGTCACCACCTGATATGGGAAAACAATGTAGG
ACAGCATTGAAATAGAATAAGCGGCGTCGCAAGAGCAGAT
AAGTTGCATATGTAACAATAGAAGLGCTAACGCAG;GCA-A
ACCGAAACACAAGCAGCAGAAACGAAATCGAGAACCAGTC
GCGCC*CATCGACGC
AGCAGTT~-GAAGTACAACAGTAACC
ACACAGTAATCAAAGAAATCGCTTCCAAAGGCGc~AATrT
AGATAGTTACCATCGAATGAACCCTGTCGTAAACATCGTA
GAATGCGAGTTGTAAATTAACTCCAGACCCAGCTGAACAC
GCCCCGTTTCACTAACT(GGATCGGGGTGGCCGCAACGGGG
CTGCAGATACCCGTGCAAAAAAGTGAIGCTGCCGACGCGCG
AAACAAkCGACGCGAAGTCGAAATAGAAGTAACTATCGCA
TTAGAGC~-TACGTCATTTCCCATCATGCGACCTACTGGG
GGGTCAGAAAAAACATCCTACGCTCGCGGAAAAGTCGGA
TCGGAAGTGGACCATCGACGGAAACGCACTCTTTGCACTC
GAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGACCGJGAJACTGACCTACACCATAGAT
TTCGCCGCCAAGCAGGGAAACGGCAAATCGACATTTGTCGCCAGACATGTCGCCTGGCCGCCGCCr.T ATAGCGTGAAGrAGCTACGGTCGCTTCACACGGAGCGTCC
CTGTTTTGGAAGCAGATGCGAGGGAQGAACTACGAAGCTT
GGCCTTGCCGCCAGACTCGAGCACCACCACCCCACCAC
'QA
1 2MKHP'PSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNGQEING *51 *FKAGETIYDI DEDGTITIUW ATAADVEADD F(GLGLAG(VV TNLTKTVNEN 101 KQNVDAKvKA AEsEiEKLTT ICLADfTDAAI.A DTDAALDATT NALNKLGENI 151 TTFAEETKTN IVI(IDEKLEA VAflTVDKHAE AFNDIADSLD ENTKADFAv 201 KTANEAXQTA EETKQNVDAX( VKAAETAAGK AEAAAGTANT AAflKAEAVAA 251 KVTDIKAflIA TNKDNIAIU(A NSAflVYTREB SDSKWVRIDG LNATTEKLDT 301 RLASABKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG 351 GSGGGGVAAD IGAGLAflALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ 401 GARKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ 451 SHSALTAPQT EQIQDSEHSG 1MVARQFRI GDIAG=HSF DKLPEGGRAT 501 YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EffLASPELNV DLAAADI:KPD 551 GKRHAVISGS VLYNQABKGS YSLGXFGGKA QEVAGSAEVK TVNGIRHIGL 601 AAKQLEHHHH HH* 96lcL-983
ATAAATTCTCAGATACCGCTCTGCCTCGACGGATGACAAA
GAGCAGTAAACGCCGGCATCGTCTCAAGCAGATACGTCA
GCGAAACTTCAATAGAAGCCATACAAAAGACGACGTTGAC
r-CATTAGTTGTTAAAGCTATACGCAACGCAGAAAAAACT
GAGCAGAAGTCGACGATGAATACACAGTGAAATAGCCTAC
GAATAGCCCGAGACACAGCTAAAATGAAATTAGCTTCGAA
ACAGCATTGAAATAGAATAAGCTGTAACTGCACTCG~.AT
AAGTTGCATATGTAhCAATAGAAGACGCMCGCAGACAAA ACGCAGACAAAACTGTCAATAAGTCGACGACGCArCGACrC
GCGCCGTAATCGCAAGCGACGTGTCAATACAACAGTAACC
ACACAGTAAMTAAZACACOGCAGGAACGGAATTAACAT~T
AGATAGTTACCATCGAATGAACCCTGTCGTAAACATCGTA
GAATGCGAGTTGT"cGGcGCTCCAGACCCAGCTGAACAC
GCCCCGTTTCACTAACTGTGTCGWGCCATCGGCGCTATC
GGGTCGTTCGACAAGGACA~rCAACWACGACTCCGTTAGA GAGGAA AAr.AGTTTCGTWATAGTC.TAAACGGTCAAACA
GCCCCCGACGAACGGCTCAACCATAGAAAGATGTACTAAC
GCATACCGCAAAGcc-aTGGTGGAiGc~~ArGhcGc.crAAc WO 01/64920 PCT/IBOI/00420 -34- TTCCACGAGCGAAACCrcAA~cAAT~AA~TTCGGAAGGAGA
GCCTAGCGGCGAAAATAGTCTTGCAGGCGTTGGCGACAGC
ACGGATATCCGCCACGTAAAGAA-ATCGGACACATCGATTTGGTCTCCCATATT1A'rTGGCGGGCGTTCCGTGGCGGC
AGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAAGATGAAACCAAGAACGAAATGATG
GTGACACGATCTGTAGTGCACGGCTCCTGCAACGTTGAAC
TCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTA1'rCC GGCGGTGATAAAACAGACGAGGGTATICCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACAC6CGTAAT
AAAAACATGCTTTTCATCTTT'TCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTAT
GAAAAAGACGCTCAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGAGTTCA"CGGATGTAT
GGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTrGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCC
TATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCG.ATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACC
GGAGCGTTCGTCGATCCTGTAGACAACTCTCAGTCGCAGC
CAGGACATCGGTGCAGTrCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCGGATGCGGGTAAGGCCATAACGGACCC GCGTCCTTTrCCGTTCGGCGACTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTT'CCGTAACGACATT
TCAGGCACGGGCGGCCTGATCAAAAAGGCGGCAGCCAACTGCAACTGCACGGACACACCTATACGGGCAAACC
ATACAGCGTGTGGTTCGACAAACGTTCCTGACA-GTCCGT
TATAACGGGGCGGCATCCGGCGGCACCTGAACACGAGGCATTGTCTATCTGGCGATACCGACAATCCGGCGCA
AACGAAACCGTACACATCAAAGQCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACJACACGTTTGGGCAAACTGCTG
AAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCGGGGCAGGCTATCTCAACAGT
ACGAGCTTCCTCGGGCCAATGGCGATTCTCTAAAACAACA
GGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGT
CGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAACACGCCGTAGAACAGGGC
GGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAACTGCG
GCAGCCGACCGCACAGATATGCCGGrGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCG
ATGCCGCCGACGGTGTACGCATCTACAGTCTCGCCGCTACCGTCTATGCCGCAGTACCGCCGCCCATGCGAT
ATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGTCTGCGCGTCATCGCGCAAACC
CAACAAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGG CAGTACCCAAACCGTCGGCATTGCC GCGAAAACCQ7GCGAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAJACAGTGCAAAT
GCAAAAACCGACAGCATTAGTCTGTTCAGGCATACGGCACGATGCGGGCGATAGGCTATCTAGCCTGTTC
I'CCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACG
CTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTG
CGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAA
r.GCACGCTGGTCGGACTCGCGGGTCTGAGrCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCACGGCGGGC
GTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACG
GGGAGATTCGAACGCGTGCGCTGCCGTTGATGCAGCGACG
TGGCACGTTACAGCTACGCCGGTTCCAACAGTACGGCAACCACAGCGGACGATCGGCGTAGGCTACCGGTTCTGA
CTCGAG
1 WCHFPSKVLT TAILATFCSG ALAATNDDDV KI(AATVAIAA AYNNGQEING 51 P1(AGBTIYDI DEDGTITKKD ATAAflVFADD F'KGLGLKKVV TNLTKTVNEN 101 I{QNVDAKVYA AESEIEKLTT KLADTDAAI.A DTDAALDATT NALNKLGENI 151 TTPAEETKTN IVKIDBKLEA VADTVDRHAE AFNDIAflSLD ETNTKADEAV 201 KTANEM<QTA EETKQNVDAK VKAAETAAGK AEAAZAGTANT AAflKAEAVAA 251 KVTDflCADIA TNKDNIACKA NSAflVYTREE SDSKFVRIDG LNATI'EKLDT 301 RLASAEKSIA DHDT1U.NGLD KTVSDLRICET RQGLAEQA~A SGLjFQPYNVG 351 GSGGGGTSAP DFNAGGTGIG SNSRATTAKS AAVSYAGIIN EMCKDRSLC 401 AGRflDVAVTD RDAIIAPPP NLHTGDFPNP NDAYKNLINL KPAIEAGYTG 451 RGVEVGIVUI' GESVGSISFP ELYGEIKEHGY NENYKNYTAY MRKEAPEDGG 501 GKDIEASFDD EAVIETEAKP TDIRHVKCEIG HIDLVSHIIG GRSVDGRPAG 551 GIAPDATLHI M1NTNDETIQNE bMAAIRNAW VKLGERGVRI VNNSPGTTSR 601 AGTAflLFQIA NSEEQYRQAL LDYSGGDKTD EGIRLMQQSD YGNLSYHIRN 651 KNMLFIFSTG NDAQAQPNTY ALLPFYEKDA QKGIIrWAGV DRSGEKPKRE 701 MYGEPGTEPL EYGSNHCGIT AMWCLSAPYE ASVRFIRTNP IQIAGTSFSA 751 PflPIGTAALL LQKYPWMSND NLRTTLLTTA QDIGAVGVDS KFGWGLLDAG 801 KAIMN'GPASFP FGDrTADTKG TSDIAYSFRN DISGTGGLIK ICGGSQLQLHG 851 NNTYTGKTII flGGSLVLYGN NKSDMRVETK GALIYNGAAS GGSLNSDGIV 901 YLAflTDQSGA NETVHIKGSL QLDGKGTLYT RLGKLLKVDGTAIIGGKLYM 951 SARGKGAGYL NSTGRRVPFL SAM(IGQDYS FP1'NIETDGG LILASLDSVEK 1001 TAGSEGDTLS YWVRRGNAAR TASAAAHSAP AGILKHAVEOG GSNLENL!4VE 1051 LDASESSATP ETVETAAADR TDMPGIRPYG ATFRAAAAVQ HANAAflGVRI 1101 FNSLAATVYA DSTAAHAflMQ GRRLKAVSDG LDH1NGTGLRV IAQTQQDGGT 1151 WEQGGV33GKM RGSTQTVGIA AKTGE1NTTAA AThGMGRSTW SENSANAKTD 1201 SISLFAGIRH DAGDIGYLKG LFSYGRYKNS ISRSTGADEH AEGSVNGTIM4 651251 QLGAL0GVI'V PEAATGDLTV EGGLRYDLLK QDAPAEKGSA LGWSGNSLTE 651301 GTLVGLAGLK LSQPLSDKAV LPATAGVERD LNGRDYTVTG GFTGATAATG 1351 KTGAPINMPHT RLVAGLGADV EPGNGWNGLA RYSYAGSKQY GNHSGRVGVG 1401 YRF* WO 01/64920 PCT/IB01/00420 It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. For instance, the use of proteins from other strains is envisaged see WOOO/66741 for polymorphic sequences for ORF4, ORF40, ORF46, 225, 235, 287, 519, 726, 919 and 953].
EXPERIMENTAL DETAILS Cloning strategy and oligonucleotide design Genes coding for antigens of interest were amplified by PCR, using oligonucleotides designed on the basis of the genomic sequence of N. meningitidis B MC58. Genomic DNA from strain 2996 was always used as a template in PCR reactions, unless otherwise specified, and the amplified fragments were cloned in the expression vector pET21b+ (Novagen) to express the protein as C-terminal His-tagged product, or in pET-24b+(Novagen) to express the protein in 'untagged' form AG 287K).
Where a protein was expressed without a fusion partner and with its own leader peptide (if present), amplification of the open reading frame (ATG to STOP codons) was performed.
Where a protein was expressed in 'untagged' form, the leader peptide was omitted by designing the 5'-end amplification primer downstream from the predicted leader sequence.
The melting temperature of the primers used in PCR depended on the number and type of hybridising nucleotides in the whole primer, and was determined using the formulae: Tmi 4 2 (tail excluded) Tm2 64.9 0.41 GC) 600/N (whole primer) The melting temperatures of the selected oligonucleotides were usually 65-70 0 C for the whole oligo and 50-60 0 C for the hybridising region alone.
Oligonucleotides were synthesised using a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2.0ml NH 4 OH, and deprotected by 5 hours incubation at 56 0 C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were centrifuged and the pellets resuspended in water.
Sequences Restriction site fu (961)- Fwd CGCGGATCC -GGAGGGGGTGGTGTCG Baml WO 01/64920 WO 0164920PCT/IB01/00420 741(MCS8)-His Rev CCCG-CTC-Q-TTGC1TGGCGGCAAGGC XhoI Pu (961 ).983-His Fwd CGCDA=~- GGCGGAGGCGGCACITr BamlH Rev CCCGCgCGAG-GAACCGGTAGCCTACG XhoI fu (961). 0rf46.1. Fwd 'CGCGGATCCGGTGGTGGTGGT- BamHl His iTCAGATrTGGCAAACGATTC Rev CCCGCTCGAG-CGTATCATATrTCACGTGC XhoI fu (961 Fwd CGCGGATCC -GGAGGGGGTGGTGTCG BaniH1 741(MC58) Rev CCCG-CTCGAG-TTATTGCTTGGCGGCAAG XhoI fu (961c-L )-983 Fwd CGCGGzA.TCC -GGCGGAGGCGGCAcTr BamIH Rev CCCGCIAG-TCAGAACCGGTAGCCTAC XhoI Pu (961c.L)- Fwd CGCaQAGGTGTGGTGGT- BamHl 0rf46.1 TCAGAT'ITGGCAAACGATI2C Rev CCCG=TGAG-'TTACGTATCATA ITrCACGTGC XhoI fu-(AG287)-919. Fwd CGCGQATCCGGTGGTGGTGGT- BaniHI His CAAAGCAAGAGCATCCAAACC Rev CCCAAGC'ITTCGGGCGGTATTCGGGC'rrC HindMl fu-(AG287)-953. Fwd CGCGGUATCCOGTGGTGGTGGT- BamMi Is IGCCACCTACAAAGTGGAC GCCCAA=I-TTGTrGGCTGCCTCGAT H-indu~ fu-(AG287)-M6- Fwd CGCGG;ATCCGGTGGTGGTGG.T-ACAAGCGACGACG BamHI His Rev GCCCAAGCTT-CCACTCGTAAITGACGCC Hlindli Fwd CGCGGATCCGGTOGTGGTGGT- BaniHI 0rf46.1-His __TCAGATITGOCAAACGAITC Rev CCCAAGCTT-CGTATCATATTrCACGTGC. indIll fu-(AG287-919). Fwd CCCAAGCTlGGTGGTGGTGGTGGT- HindIu Orf46.1-His __TCAGATITGGCAAACGA112C Rev CCCGCTCGAG-CGTATCATATflTCACGTGC XhoI fu-(AG287- Fwd CCCAAGCTGGTGGTGGTGGTGGT- Hindll 0rf46.1)-919-His __CAAAGCAAGAGCATCCAAACC C C C OCrC GA--CGGG CG G TAT C G GG C TT XboI fu AG287(394.98)- Fwd CGCGGATCC GCTAGC-CCCGATGTTAAATCGGC NheI Rev CGG-GgAT-C-ATCCTGCTCrTrrrGCCGG BaniHl fu Orfl-(Orf46.1)- Fwd CGCG3GATCCGfrAGC-GGACACAc'TATrcGocATc Nhei His -Rev CGCGCATC-ccAGcGGTAGccTAATTGAT Pu (Orfl)-Orf46.1- Fwd CGCGQ=CGGTOGTGGTOGT- BainHI His TCAGA TI2GGCAAACGATTC Rev CCCAAGCTT-CGTATCATATITCACGTGC HindI Pu (919)-Orf46.1- Fwdl GcGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAG Sail His Fwd2, GGAGGCACrGGATCCTCAGATI'GGCAAACGATrC ICCCg.CI'CGAG-CGTATCATATTCACGTGC XhoI Fu (orf46)-287-EHis Fwd CGGGGQATCGGGGGCGGCGGTGGCG BaniHl Rev CCCAAGCIATCCrGCTCTMTI2GCCGOC Hindifi Pu (orf46)-919-His Fwd CGCGGATCGGTOGTGGTGGTCAAkGCAAGACCATCCA BainHI __AACC CCCA&GCTTCGGGCGGTATTCGGGCTTC HindIfI WO 01/64920 WO 0164920PCT/IBOI/00420 Fu (orf46-919)- Fwd CCCCAA&=GGGGGCGGCGGTGGCG HindIl 287-His Rev CCCG(ZECOAUATCCrGCTCTTIGCCGGC XhOI Fu (orf46-287)- Fwd CCCMAflGGTGGTGGTGGTGGTCAAAGCAAGAGCAT HindM 919-His CCAAACC Rev CCCGCTCGAGCGGGCGGTATTCGGGCIT XhoI (AG741 )-961c-His Fwdl GGAGQCACTGOATCCGCAGCCACAAACGACGACGA Xho0I Fwd2 GCGGCC~-GGTGGGGCGG(CACTGGATCCGjCAG Rev CCCGC'FCGA-G-ACCCAGC'1TGTAAGGTrG Xho0I (AG741 )-961-His Fwdl GGAGGCACrGGATCCGCAGCCACAAACGACGACGA XhoI Fwd2 GCGGC!CrCGAG-GGTGGCGGAGGCACTGGATCCGCAG Rev CCGCTGAG-CCACTCGTAATTGACGCC Xhol (AG741 )-983-His Fwd GCGGCCQAg- Xho0I __GGATCCGGCGGAGGCGGCAC'rc'GCG Rev CCCG=aA-GAACCGGTAGCCTACG XhoI (AG741 )-orf46.1- Fwdl GGAGGCACrGGATCCTCAGA ITIGGCAAACGAITC Sail His Fwd2 GCGGCO-ICQAGGTGGCGGAGGCACTGGATCCTCAGA Rev CCCGCTCGAG-CGTATCATATITCACGTGC XhoI (AG983)- Fwd QCGGCCTCGAG-GGATCCGGAGGGI3GTGGTGTCGCC XhoI 741(MC58) -His Rev CCCG(ZE Q-ITGCTTGGCGGCAAG X11oI (AG983).961c-His Fwdl GGAGGCACTGGATCCGCAGCCACAAACGACGACGA XlioI GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG CCCG=rGAG-ACCCAGCrI2GTAAGGT2G XhoI (AG983)-961-His Fwdl GGAGGCACTGGATCCGCA.GCCACAAACGACGACGA XhoI Fwd2 GCGGC-CTA-GGTGGCGGAGGCACTGGATCCGCAG CCCGCTCOCAG-CCACTCGTAA ITGACGCC XhOI (AG983)- 0r46.1- Fwdl GGAGGCACTGGATCCTCAGATTTGGCAAACGAT2C Sail His Fwd2 GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGA Rev CCCGCTCGAG-CGTATCATATITCACGTGC XhoI *This primer was used as a Reverse primer for all the C terminal fusions of 287 to the His-tag.
§Forward primers used in combination with the 287-His Reverse primer.
NB All PCR reactions use strain 2996 unless otherwise specified strain MC58) In all constructs starting with an ATG not followed by a unique NheI site, the ATG codon is part of the NdeI site used for cloning. The constructs made using NheI as a cloning site at the end all those containing 287 at the N-terminus) have two additional codons (GCT AGC) fused to the coding sequence of the antigen.
Preparation of chromosomal DNA templates NMmeningitidis strains 2996, MC58, 394.98, 1000 and BZ232 (and others) were grown to exponential phase in lO0mi of GC medium, harvested by centrifugation, and resuspended in Sail buffer. (20% w/v sucrose, 50mM Tris-HC1, 50mM EDTA, pH 8 After 10 minutes incubation on ice, the bacteria were lysed by adding l1ini of lysis solution (50mM NaCI, 1% Na-S arkosyl, 5Opg/nil Proteinase and the suspension incubated at 37 0 C for 2 hours. Two WO 01/64920 PCT/IB01/00420 -38phenol extractions (equilibrated to pH 8) and one CHC1 3 /isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes of ethanol, and collected by centrifugation. The pellet was washed once with ethanol and redissolved in 4.0ml TE buffer (10mM Tris-HCI, lmM EDTA, pH The DNA concentration was measured by reading OD 2 6 o.
PCR Amplification The standard PCR protocol was as follows: 200ng of genomic DNA from 2996, MC581000, or BZ232 strains or lOng of plasmid DNA preparation of recombinant clones were used as template in the presence of 40pM of each oligonucletide primer, 400-800 pM dNTPs solution, Ix PCR buffer (including 1.5mM MgC1 2 2.5 units TaqI DNA polymerase (using Perkin-Elmer AmpliTaQ, Boerhingher Mannheim Expand T Long Template).
After a preliminary 3 minute incubation of the whole mix at 95 0 C, each sample underwent a two-step amplification: the first 5 cycles were performed using the hybridisation temperature that excluded the restriction enzyme tail of the primer (Tml). This was followed by 30 cycles according to the hybridisation temperature calculated for the whole length oligos (Tm2).
Elongation times, performed at 68 0 C or 72 0 C, varied according to the length of the Orf to be amplified. In the case of Orfl the elongation time, starting from 3 minutes, was increased by seconds each cycle. The cycles were completed with a 10 minute extension step at 72 0
C.
The amplified DNA was either loaded directly on a 1% agarose gel. The DNA fragment corresponding to the band of correct size was purified from the gel using the Qiagen Gel Extraction Kit, following the manufacturer's protocol.
Digestion of PCR fragments and of the cloning vectors The purified DNA corresponding to the amplified fragment was digested with the appropriate restriction enzymes for cloning into pET-21b+, pET22b+ or pET-24b+. Digested fragments were purified using the QIAquick PCR purification kit (following the manufacturer's instructions) and eluted with either HzO or 10mM Tris, pH 8.5. Plasmid vectors were digested with the appropriate restriction enzymes, loaded onto a 1.0% agarose gel and the band corresponding to the digested vector purified using the Qiagen QIAquick Gel Extraction Kit.
WO 01/64920 PCT/IB01/00420 -39- Cloning The fragments corresponding to each gene, previously digested and purified, were ligated into pET21b+, pET22b+ or pET-24b+. A molar ratio of 3:1 fragment/vector was used with T4 DNA ligase in the ligation buffer supplied by the manufacturer.
Recombinant plasmid was transformed into competent E.coli DH5 or HB 101 by incubating the ligase reaction solution and bacteria for 40 minutes on ice, then at 37 0 C for 3 minutes.
This was followed by the addition of 80 0 pl LB broth and incubation at 37 0 C for 20 minutes.
The cells were centrifuged at maximum speed in an Eppendorf microfuge, resuspended in approximately 200pl of the supernatant and plated onto LB ampicillin (100mg/ml) agar.
Screening for recombinant clones was performed by growing randomly selected colonies overnight at 37 0 C in 4.0ml of LB broth 100lg/ml ampicillin. Cells were pelleted and plasmid DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions. Approximately lpg of each individual miniprep was digested with the appropriate restriction enzymes and the digest loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1kb DNA Ladder, GIBCO). Positive clones were selected on the basis of the size of insert.
Expression After cloning each gene into the expression vector, recombinant plasmids were transformed into E.coli strains suitable for expression of the recombinant protein. lpl of each construct was used to transform E.coli BL21-DE3 as described above. Single recombinant colonies were inoculated into 2ml LB+Amp (100lpg/ml), incubated at 37 0 C overnight, then diluted 1:30 in 20ml of LB+Amp (100lg/ml) in 100ml flasks, to give an OD 6 0 o between 0.1 and 0.2.
The flasks were incubated at 30 0 C or at 37°C in a gyratory water bath shaker until OD 6 00 indicated exponential growth suitable for induction of expression (0.4-0.8 OD). Protein expression was induced by addition of 1.0mM IPTG. After 3 hours incubation at 30°C or 37 0 C the OD 60 0 was measured and expression examined. 1.0ml of each sample was centrifuged in a microfuge, the pellet resuspended in PBS and analysed by SDS-PAGE and Coomassie Blue staining.
Purification of His-tagged proteins Various forms of 287 were cloned from strains 2996 and MC58. They were constructed with a C-terminus His-tagged fusion and included a mature form (aa 18-427), constructs with WO 01/64920 PCT/IB01/00420 deletions (Al, A 2, A3 and A4) and clones composed of either B or C domains. For each clone purified as a His-fusion, a single colony was streaked and grown overnight at 37 0 C on a LB/Amp (100 pg/ml) agar plate. An isolated colony from this plate was inoculated into of LB/Amp (100 pg/ml) liquid medium and grown overnight at 37°C with shaking.
The overnight culture was diluted 1:30 into 1.0 L LB/Amp (100 pg/ml) liquid medium and allowed to grow at the optimal temperature (30 or 37 0 C) until the OD 55 0 reached 0.6-0.8.
Expression of recombinant protein was induced by addition of IPTG (final concentration and the culture incubated for a further 3 hours. Bacteria were harvested by centrifugation at 800 0g for 15 min at 4 0 C. The bacterial pellet was resuspended in 7.5 ml of either cold buffer A (300 mM NaCI, 50 mM phosphate buffer, 10 mM imidazole, pH for soluble proteins or (ii) buffer B (10mM Tris-HCI, 100 mM phosphate buffer, pH 8.8 and, optionally, 8M urea) for insoluble proteins. Proteins purified in a soluble form included 287-His, Al, A2, A3 and A4287-His, A4287MC58-His, 287c-His and 287cMC58-His.
Protein 287bMC58-His was insoluble and purified accordingly. Cells were disrupted by sonication on ice four times for 30 sec at 40W using a Branson sonifier 450 and centrifuged at 13000xg for 30 min at 4 0 C. For insoluble proteins, pellets were resuspended in 2.0 ml buffer C (6 M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris- HC1, pH and treated with 10 passes of a Dounce homogenizer. The homogenate was centrifuged at 13000g for 30 min and the supernatant retained. Supernatants for both soluble and insoluble preparations were mixed with 1501Al Ni 2 -resin (previously equilibrated with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for min. The resin was Chelating Sepharose Fast Flow (Pharmacia), prepared according to the manufacturer's protocol. The batch-wise preparation was centrifuged at 700g for 5 min at 4°C and the supernatant discarded. The resin was washed twice (batch-wise) with buffer A or B for 10 min, resuspended in 1.0 ml buffer A or B and loaded onto a disposable column. The resin continued to be washed with either buffer A at 4°C or (ii) buffer B at room temperature, until the OD 2 so 0 of the flow-through reached 0.02-0.01. The resin was further washed with either cold buffer C (300mM NaCi, 50mM phosphate buffer, imidazole, pH 8.0) or (ii) buffer D (10mM Tris-HCI, 100mM phosphate buffer, pH 6.3 and, optionally, 8M urea) until OD 2 so of the flow-through reached 0.02-0.01. The His-fusion protein was eluted by addition of 700pl of either cold elution buffer A (300 mM NaC1, phosphate buffer, 250 mM imidazole, pH 8.0) or (ii) elution buffer B (10 mM Tris-HC1, 100 mM phosphate buffer, pH 4.5 and, optionally, 8M urea) and fractions WO 01/64920 PCT/IB01/00420 -41collected until the OD2s 0 indicated all the recombinant protein was obtained. 20pl aliquots of each elution fraction were analysed by SDS-PAGE. Protein concentrations were estimated using the Bradford assay.
Renaturation of denatured His-fusion proteins.
Denaturation was required to solubilize 287bMC8, so a renaturation step was employed prior to immunisation. Glycerol was added to the denatured fractions obtained above to give a final concentration of 10% v/v. The proteins were diluted to 200 jg/ml using dialysis buffer I (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, mM oxidised glutathione, 2.0M urea, pH 8.8) and dialysed against the same buffer for 12-14 hours at 4 0 C. Further dialysis was performed with buffer II (10% v/v glycerol, arginine, 50mM phosphate buffer, 5.0mM reduced glutathione, 0.5mM oxidised glutathione, pH 8.8) for 12-14 hours at 4 0 C. Protein concentration was estimated using the formula: Protein (mg/ml) (1.55 x OD2so) (0.76 x OD 26 o) Immunization Balb/C mice were immunized with antigens on days 0, 21 and 35 and sera analyzed at day 49.
Sera analysis ELISA The acapsulated MenB M7 and the capsulated strains were plated on chocolate agar plates and incubated overnight at 37*C with 5% CO 2 Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following
OD
62 0 The bacteria were let to grow until the OD reached the value of 0.4-0.5. The culture was centrifuged for 10 minutes at 4000rpm. The supernatant was discarded and bacteria were washed twice with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 1 hour at 37 0 C and then overnight at 4°C with stirring. 100l bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4°C. The wells were then washed three times with PBT washing buffer Tween-20 in PBS). 200pl of saturation buffer polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37 0 C. Wells were washed three times with PBT. 200l of diluted sera (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN 3 in PBS) were added to each well and the plates incubated for 2 hours at 37 0 C. Wells were washed three times with PBT. 100pl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37 0 C. Wells WO 01/64920 PCT/IB01/00420 -42were washed three times with PBT buffer. 100pl of substrate buffer for HRP (25ml of citrate buffer pH5, 10mg of O-phenildiamine and 10l1 of H 2 0 2 were added to each well and the plates were left at room temperature for 20 minutes. 100pl 12.5% H 2 S0 4 was added to each well and OD490 was followed. The ELISA titers were calculated abitrarely as the dilution of sera which gave an OD 49 0 value of 0.4 above the level of preimmune sera. The ELISA was considered positive when the dilution of sera with OD490 of 0.4 was higher than 1:400.
Sera analysis FACS Scan bacteria binding assay The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37 0 C with 5% CO 2 Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD620. The bacteria were let to grow until the OD reached the value of 0.35-0.5.
The culture was centrifuged for 10 minutes at 4000rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer BSA in PBS, 0.4% NaN 3 and centrifuged for 5 minutes at 4 000rpm. Cells were resuspended in blocking buffer to reach OD620 of 0.05.
100lp bacterial cells were added to each well of a Costar 96 well plate. 10lp of diluted (1:100, 1:200, 1:400) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4 0 C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200pl/well of blocking buffer in each well. 100lp of R-Phicoerytrin conjugated F(ab) 2 goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4 0 C. Cells were spun down by centrifugation at 4000rpm for 5 minutes and washed by addition of 200pl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200pl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan (Laser Power setting were: FL2 on; FSC-H threshold:92; FSC PMT Voltage: E 01; SSC PMT: 474; Amp.
Gains 6.1; FL-2 PMT: 586; compensation values: 0.
Sera analysis bactericidal assay N. meningitidis strain 2996 was grown overnight at 37 0 C on chocolate agar plates (starting from a frozen stock) with 5% CO 2 Colonies were collected and used to inoculate 7ml Mueller-Hinton broth, containing 0.25% glucose to reach an OD620 of 0.05-0.08. The culture was incubated for approximately 1.5 hours at 37 degrees with shacking until the OD620 reached the value of 0.23-0.24. Bacteria were diluted in 50mM Phosphate buffer pH 7.2 containing 10mM MgCI 2 10mM CaCI 2 and 0.5% BSA (assay buffer) at the working dilution of 105 CFU/ml. The total volume of the final reaction mixture was 50 pl with 25 pI WO 01/64920 PCT/IB01/00420 -43of serial two fold dilution of test serum, 12.5 pI of bacteria at the working dilution, 12.5. p1 of baby rabbit complement (final concentration Controls included bacteria incubated with complement serum, immune sera incubated with bacteria and with complement inactivated by heating at 56 0 C for 30'. Immediately after the addition of the baby rabbit complement, 10pl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time The 96-wells plate was incubated for 1 hour at 37 0 C with rotation. 7pl of each sample were plated on Mueller-Hinton agar plates as spots, whereas 10lp of the controls were plated on Mueller-Hinton agar plates using the tilt method (time Agar plates were incubated for 18 hours at 37 degrees and the colonies corresponding to time 0 and time 1 were counted.
Sera analysis western blots Purified proteins (500ng/lane), outer membrane vesicles (5pg) and total cell extracts derived from MenB strain 2996 were loaded onto a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150mA at 4°C, using transfer buffer Tris base, 1.44% glycine, 20% methanol). The membrane was saturated by overnight incubation at 4'C in saturation buffer (10% slimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37°C with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti- 4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.
The OMVs were prepared as follows: N. meningitidis strain 2996 was grown overnight at 37 degrees with 5% CO 2 on 5 GC plates, harvested with a loop and resuspended in 10 ml of 20mM Tris-HCI pH 7.5, 2 mM EDTA. Heat inactivation was performed at 56 0 C for minutes and the bacteria disrupted by sonication for 5 minutes on ice (50% duty cycle, output Branson sonifier 3 mm microtip). Unbroken cells were removed by centrifugation at 5000g for 10 minutes, the supernatant containing the total cell envelope fraction recovered and further centrifuged overnight at 50000g at the temperature of 4 0 C The pellet containing the membranes was resuspended in 2% sarkosyl, 20mM Tris-HCI pH 7.5, 2 mM EDTA and incubated at room temperature for 20 minutes to solubilise the inner membranes. The suspension was centrifuged at 100OOg for 10 minutes to remove aggregates, the supernatant was further centrifuged at 50000g for 3 hours. The pellet, containing the outer membranes WO 01/64920 PCT/IB01/00420 -44was washed in PBS and resuspended in the same buffer. Protein concentration was measured by the D.C. Bio-Rad Protein assay (Modified Lowry method), using BSA as a standard.
Total cell extracts were prepared as follows: N. meningitidis strain 2996 was grown overnight on a GC plate, harvested with a loop and resuspended in lml of 20mM Tris-HC1.
Heat inactivation was performed at 56 0 C for 30 minutes.
Claims (23)
1. A method for the simultaneous heterologous expression of two or more proteins of the invention, in which two or more proteins of the invention are fused.
2. The method of claim 23, in which the two or more proteins are: 919 and 287; 953 and 287; 287 and ORF46.1; ORF1 and ORF46.1; 919 and ORF46.1; (f) ORF46.1, 287 and 919; 919 and 519; and ORF97 and 225.
3. The method of claim 24, in which 287 is at the C-terminal end of protein or
4. The method of any preceding claim, in which the expression is in an E.coli host. A protein expressed by the method of any preceding claim.
6. A hybrid protein of formula NH 2 -A-B-COOH, wherein A and B are different Neisserial proteins.
7. The protein of claim 6, wherein A and B are each selected from orfl, orf4, orf25, orf46, orf83, 233, 287, 2921, 564, 687, 741, 907, 919, 953, 961 and 983.
8. The protein of claim 7, wherein A and B are each selected from ORF46.1, 287, 741, 919, 953, 961 and 983.
9. The protein of claim 8, wherein at least one of said ORF46.1, 287, 741, 919, 953, 961 and 983 is used in essentially full-length form The protein of claim 8 or claim 9, wherein at least one of said ORF46.1, 287, 741, 919, 953, 961 and 983 has a deletion.
11. The protein of claim 10, wherein A and/or B has a poly-glycine deletion
12. The protein of claim 11, wherein A and/or B is AG-287, AGTbp2, AG741, or AG983.
13. The protein of claim 10, wherein A and/or B is a truncated protein.
14. The protein of claim 13, wherein A and/or B is Al-287, A2-287, A3-287 or A4-287. The protein of claim 10, wherein a domain of A and/or B is deleted. WO 01/64920 PCT/IB01/00420 -46-
16. The protein of claim 15, wherein A and/or B is 287B, 287C, 287BC, ORF461-433, ORF46433-608, ORF46, or 961c.
17. The protein of claim 6, wherein A and B are: 919 and 287; 953 and 287; 287 and ORF46.1; ORF1 and ORF46.1; 919 and ORF46.1; ORF46.1 and 919; (g) 919 and 519; or ORF97 and 225.
18. The protein of claim 17, wherein the protein is AG287-919, AG287-953, AG287-961, AG983-ORF46.1, AG983-741, AG983-961, AG983-961C, AG741-961, AG741-961C, AG741-983, AG741-ORF46.1, ORF46.1-741, ORF46.1-961, ORF46.1-961C, 961- ORF46.1, 961-741, 961-983, 961C-ORF46.1, 961C-741, 961C-983, 961CL-ORF46.1, 961CL-741, or 961CL-983.
19. The protein of claim 8, wherein A or B is 287. The protein of claim 19, wherein B is 287
21. The protein of claim 19, wherein A is AG-287
22. The protein of claim 21, wherein B is ORF46, 919, 953 or 961.
23. The protein of any one of claims 19 to 22, wherein 287 is from strain 2996 or 394/98.
24. The protein of claim 8, wherein A is 961. The protein of any one of claims 6 to 24, wherein A and B are from the same strain.
26. The protein of any one of claims 6 to 24, wherein A and B are joined directly
27. The protein of any one of claims 6 to 24, wherein A and B are joined via a linker peptide.
28. The protein of claim 27, wherein the linker peptide is a poly-glycine linker, with the proviso that B is not a AG protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2004201216A AU2004201216A1 (en) | 2000-02-28 | 2004-03-15 | Hybrid expression of Neisserial proteins |
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| GB0004695.3 | 2000-02-28 | ||
| GB0027675.8 | 2000-11-13 | ||
| AU2004201216A AU2004201216A1 (en) | 2000-02-28 | 2004-03-15 | Hybrid expression of Neisserial proteins |
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| AU2001239478A Division AU2001239478B2 (en) | 2000-02-28 | 2001-02-28 | Hybrid expression of neisserial proteins |
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| AU2004201216A Abandoned AU2004201216A1 (en) | 2000-02-28 | 2004-03-15 | Hybrid expression of Neisserial proteins |
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- 2004-03-15 AU AU2004201216A patent/AU2004201216A1/en not_active Abandoned
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