IE950351L - Insulin percursors - Google Patents

Description

-1- 8 0 630 Insulin precursors This invention relates to biosynthetic insulin. More specifically, the invention is directed to DNA-sequences encoding biosynthetic insulin precursors and to the preparation of such insulin precursors which are convertible into 5 biosynthetic human insulin by in vitro conversion.

BACKGROUND OF THE INVENTION In the past insulin has been synthezised (from synthetic A- and B-chains) or re-synthesized (from naturally derived A- and B-chains) by combining the two chains in an 10 oxidation process whereby the 6 cysteine sulfhydryl groups of the reduced chains (4 in the A-chain, 2 in the B-chain) are converted into disulfide bonds. By this method disulfide bonds cure formed largely at random, meaning that the yield of insulin with disulfide bridges correctly positioned between cysteine 15 residues A-6 and A-ll, A-7 and B-7, and A-20 and B-19, respectively, is very low.

Following the discovery of proinsulin as a biological precursor of insulin it was observed that the A- and B-polypep-tide moieties of the linear-chain totally reduced proinsulin 20 (those moieties corresponding to the A- and B-chains of insulin, respectively) could be oxidatively combined with much less randomization of the disulfide bonds to give a substantially higher yield of correctly folded proinsulin as compared with the combination of free A- and B-chains (D.F. Steiner et al.: 25 Proc.Nat.Acad.Sci. 6£ (1968), 622). Albeit high yields were obtained only at proinsulin concentrations too low to make the process feasible on a preparative scale, the function of the C-(i.e. connecting peptide) moiety of the B-C-A polypeptide sequence of proinsulin. namely that of.bringing the 6 cysteine 30 residues into spatial positions favorable for correct oxidation into proinsulin, was clearly demonstrated. 8063G The proinsulin formed may function as an iji vitro precursor of insulin in that the connecting peptide is removable by enzymatic means (W. Kemmler et al.: J.Biol.Chem. 24 6 (1971), 6786) .

Subsequently it has been shown that proinsulin-like compounds having shorter linking moieties than the C-peptide and flanked at both ends by specific enzymatic or chemical cleavage sites (the so-called miniproinsulins (A. Wollmer et al., Hoppe-Seyler's Z. Physiol.Chem. 355 (1974), 1471 - 1476 10 and Dietrich Brandenburg et al., Hoppe-Seyler's 2.

Physiol.Chem. 354 (1973), 1521 - 1524)) may also serve as insulin precursors.

Endeavours to provide biosynthetic insulins, particularly that identical to the human species, have followed the 15 same strategic pathways as those to synthetic insulin. The insulin A- and B-chains have been expressed in separate host organisms, isolated therefrom and then combined as described supra (R.E. Chance et al.; Diabetes Care £ (1982), 147). Microorganisms have been transformed with cloning vectors encoding 20 preproinsulin or proinsulin which may be secreted as such (W.

Gilbert et al.: European Patent Publ. No. 6694) or accumulated intracellularly as hybrid gene products (D.V. Goeddel et al^: European Patent Publ. No. 55945). The miniproinsulin pathway has also been attempted (D.V. Goeddel, supra).

Procuring the A- and B-chains in separate fermenta tion processes followed by combination of the chains is inherently impractical. The dual fermentation inconvenience may be overcome by choosing the proinsulin or miniproinsulin strategy. However, the use of a proinsulin as the biosynthetic 30 insulin precursor may entail certain disadvantages. The proinsulin, whether excreted into the fermentation liquid as such or accumulated intracellularly in the host organism, possibly as a hybrid gene product, is likely to contain substantially randomized disulfide bonds. The refolding of such "scrambled" 35 products into correctly folded proinsulin may be conducted either directly (H.-G. Gattner et al.: Danish Patent Application No. 4523/83) or via the single chain hexa-S-sulfonate (F.B. Hill: European Patent Publ. No. 37255). The refolding process usually entails some degree of polymerization and hence the inconvenience of using laborious purification steps during recovery.

In addition, insulin precursors of the proinsulin 5 type are prone to undergo enzymatic degradation, either within the host cells or following its excretion into the fermentation broth. In yeast it has been shown that human proinsulin is particularly sensitive to enzymatic cleavages at the two dibasic sequences (Arg31-Arg32 and Lys64-Arg65). Apparently 10 these cleavages occur before the establishment of the S-S bridges, resulting in the formation of C-peptide, A.-chain and B-chain.

OBJECT OF THE INVENTION AND SUMMARY THEREOF The object of the present invention is to circumvent these disadvantages by devising biosynthetic insulin precursors which are generated largely with correctly positioned disulfide bridges between the A- and B-moieties and, furthermore, substantially more resistant to proteolytic degradation than the biosynthetic insulin precursors known heretofore.

A single chain insulin precursor consisting of a Bl 329 shortened insulin B-chain from Phe to Lys continuing into a Al A21 complete A-chain from Gly to Asn , B(l-29)-A(l-21), is known (Jan Markussen, "Proteolytic degradation of proinsulin and of the intermediate forms",: Proceedings of the Symposium on Proinsulin, 25 Insulin and C-Peptide, Tokushima, 12 - 14 July, 1978, Editors: S. Baba et al.). This insulin precursor B(1-29)-A(1-21) is prepared by a semisynthetic process from porcine insulin. First the form insulin B(l-29) and A(l-21) chains were prepared and coupled to/a linear peptide B(1-29)-A(1-21). This compound in the hexathiol 30 form was oxidized in vitro rendering the single chain des-(B30) insulin molecule.

The present invention is based on the surprising discovery that the above single chain insulin precursor B(l-29)-A(l-21) and derivatives thereof with a bridging chain connecting 35 the carboxyl terminus of the B(1-29)-chain with the amino terminus of the A(1-21)-chain are expressed in high yields and with correctly positioned disulfide bridges when yeast strains transformed with DNA-sequences encoding such insulin precursors are cultured.

According to a first aspect of the present invention 5 there is provided human insulin precursors of the formula B(l-29)-Xn-Y-A(l-21) I wherein X^ is a peptide chain i with n amino acid residues, Y is Lys or Arg, n is an integer from 0 to 33, B( 1-29) is a Bl 009 shortened B-chain of human insulin from Phe to Lys and A(l-21) is the A chain of human insulin, with the proviso that the peptide chain -X -Y- does not contain two adjacent basic amino acid residues (i.e. Lys and Arg).

Preferred insulin precursors of the above formula I are compounds 15 with a relative short bridging chain between the B(1-29)- and the A(l-21)- chain.

N is preferably 1-33, more preferably 1-15, more preferably 1-8 or 1-5 and most preferably 1-3 or 1-2. X may preferably be 20 selected from the group consisting of Ala, Ser and Thr, the individual X*s being equal or different- Examples of such preferred compounds are B(1-29)-Ser-Lys-A<1-21) and B(l-29)-Ala-Ala-Lys-A(l-21).

There is provided a replicable expression vehicle capable of expression of a DNA-sequence comprising a sequence encoding the insulin precursors of formula I in yeast.

The expression vehicle may be a plasmid capable of replication in the host microorganism or capable of integration 30 into the host organism chromosome. The vehicle employed may code for expression of repeated sequences of the desired DNA-sequence, each separated by selective cleavage sites.

There is provided a process for producing insulin precursors of 35 formula I in yeast wherein a transformant yeast strain including at least one expression vehicle capable of expressing the insulin precursors is cultured in a suitable nutrient medium followed by isolation of the insulin precursors.

Preferred novel insulin precursors are B(l-29)-Ser-Lys-A(l-21) and B(1-29)-Ala-Ala-Lys-A(1-21).

There is provided a yeast strain transformed with an expression 10 vehicle capable of expressing a DNA-sequence comprising a sequence encoding the above insulin precursors in yeast.

The insulin precursors may be expressed with additional protein proceeding the insulin precursor. The additional protein may have the function of protecting the insulin precursor 15 against, e.g. in vivo degradation by endogeneous enzymes or of providing information necessary to transport the desired protein into the periplasmic space and finally across the cell wall into the medium.

The additional protein contains a selective cleavage 20 site adjacent to the N-terminal of the B(1-29)-chain of the insulin precursors enabling subsequent splitting off of the additional protein either by the microorganism itself or by later enzymatical or chemical cleavage.

Accordingly there is disclosed a DNA-25 sequence encoding the above insulin precursors and further comprising an additional DNA-sequence positioned upstream to the sequence encoding the insulin precursors and encoding an additional amino acid-sequence containing a selective cleavage site adjacent to the N-terminal of the B(1-29)-chain of the insulin 3 0 precursors.

According to a preferred embodiment of the present invention the additional amino acid sequence comprises at least one basic amino acid adjacent to the N-terminal of the B(l-29)-chain of the insulin precursor.

When the insulin precursor is expressed in yeast the additional amino acid-sequence may contain two basic amino acids (e.g. Lys-Lys, Arg-Arg, Lys-Arg or Arg-Lys) adjacent to N-terminal of the B(1-29)-chain of the insulin precursor, yeast 5 being able to cleave the peptide bond between the basic amino acids and the precursor. Also a Glu-Ala or Asp-Ala cleavage site adjacent to the desired protein enables separation of the additional amino acid sequence by the yeast itself by means of a dipeptidase enzyme produced by the yeast.

The insulin precursors may be secreted with an amino acid-sequence linked to the B(1-29)-chain of the precursors provided that this amino acid sequence contains a selective cleavage site adjacent to the B(l-29)-chain for later splitting of the superfluous amino acid sequence. If the insulin precursors 15 do not contain methionine cyanogen bromide cleavage at methionine adjacent to the desired protein would be operative. Likewise, arginine- and lysine-cleavage sites adjacent to the desired protein enables cleavage with trypsinlike proteases.

For secretion purposes the DNA-sequence encoding the 20 insulin precursors may be fused to an additional DNA-sequence coding for a signal peptide. The signal peptide is cleaved off by the transformant microorganism during the secretion of the expressed protein product from the cells ensuring a more simple isolation of the desired product.The secreted product may be the 25 insulin precursor or may contain an additional N-terminal amino acid-sequence to be removed later as explained above.

Secretion may be provided by including in the expression vehicle the yeast MFal leader sequence (Kurjan, J. and Herskowitz, I., Cell 3K), (1982), 933 - 943) and according to a 30 further preferred embodiment of the present invention the additional amino acid-sequence positioned upstream to the sequence encoding the insulin precursors comprises the yeast MFal leader coding sequence or part thereof.

The expression of the desired DNA-sequence will be 35 under control of a promoter sequence correctly positioned to the DNA-sequence encoding the desired protein product to result in expression of the desired protein in the host organism. Preferably a promoter from a gene indigeneous to the host organism may be used. The DNA-sequence for the desired protein will be followed by a transcription .terminator sequence, preferably a terminator sequence from a gene indigeneous to the host organism. If yeast is used as host organism the promoter and terminator sequences 5 are preferably the promoter and terminator of the triose phos-phase isomerase (TPI) gene, respectively.

Other promoters may be utilized such as the phosphogly-cerate kinase (PGKl)- and the MFa1-promoter.

There is also disclosed a method for a method for preparing human insulin by which a yeast strain is transformed with a replicable expression vehicle comprising a DNA-sequence encoding the insulin precursors of the above formula I, the transformed yeast strain is cultured in a suitable nutrient medium, the insulin precursors are recovered from the culture 15 medium and converted in vitro into human insulin.

The insulin precursors according to the present invention may be converted into mature human insulin by transpep-tidation with an L-threonine ester in the presence of trypsin or a trypsin derivative as described in the specification of Danish 20 patent application 574/80 (the disclosure of which is incorporated by reference hereinto) followed by transformation of the threonine ester of human insulin into human insulin by known processes. See also U.K. Patent Publication No. 20669502B.

If the insulin precursors are secreted with an additional amino acid sequence adjacent to the N-terminal of the B(l-29)-chain such amino acid sequence should either be removed in vitro before the transpeptidation or should contain at least one basic amino acid adjacent to the N-terminal of the B(l-29)- chain as trypsin will cleave the peptide bond between the basic Bl amino acid and the amino group of Phe during the transpeptidation.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings illustrate a preferred embodiment of the present invention.

Fig. 1 illustrates the preparation of plasmid pMT344, fig. 2 illustrates the preparation of plasmid pMT475, fig. 3 illustrates the preparation of plasmid pMT212, fig. 4 illustrates the preparation of plasmid pMT4 79 5 fig. 5 .illustrates the preparation of plasmid ptfT319, fig. 6 illustrates the preparation of plasmid pMT598, fig. 7 illustrates the preparation of plasmid pMT610, fig. 8 illustrates the preparation of plasmid pT5, and 10 fig. 9 illustrates the preparation of plasmid pMT639.

In the drawings and part of the following description the expression B' is used in stead of B(l-29) and A in stead of A(l-21). Accordingly the expression B'A is equivalent to the expression B(1-29)-A(1-21).

DETAILED DESCRIPTION 1. Preparation of a gene coding for human proinsulin B-C-A Total RNA purified (Chirgwin, J.M. Przybyla, A.E., McDonald, R.J. & Rutter, W.J., Biochemistry 18, (1979) 5294 -5299) from human pancreas was reverse transcribed (Boel, E., 20 Vuust, J., Norris, F., Norris, K., Wind, A., Rehfeld, J.F. & Marcker, K.A., Proc.Natl.Acad.Sci. USA 80, (1983), 2866 - 2869) with AMV reverse transcriptase and d(GCTTTATTCCATCTCTC) as 1. strand primer. After preparative urea-poly aery lamide gel purification of the human proinsulin cDUA, the second strand-was* 25 synthesized on this template with DNA polymerase large fragment and d(CAGATCACTGTCC) as 2nd strand primer. After Si nuclease digestion the human proinsulin ds. cDNA was purified by poly aery 1 ar.o de gel electrophoresis, tailed with terminal transferase and cloned in the PstI site on pBR327 (Sorberon et 30 al^.. Gene 9, (1980), 287 - 305) in E.. coli. A correct clone harbouring a plasmid cpntaining a gene encoding human proinsulin B-C-A was identified from the recombinants by restriction endonuclease analysis and confirmed by nucleotide sequencing (Maxam, A., & Gilbert, W., Methods in Enzymology, 65 (1980), 499 = 560. Sanger, F.,"Nicklen, S. & Coulson, A.R., Proc.Natl.Acad.Sci. USA 74, (1977), 5463 - 5467). 2_. Preparation of genes coding for precursors of human insulin. 5 The gene encoding B(1-29)-A(1-21) of human insulin was made by site specific mutagenesis of the human proinsulin sequence with a 75bp in frame deletion in the C-peptide coding region inserted into a circular single stranded M-13 bacteriophage vector. A modified procedure (K. Norris et al., 10 Nucl.Acids.Res. 11 (1983) 5103 - 5112) was used in which a chemically synthesized 19-mer deletion primer was annealed to the Ml3 template. After a short enzymatic extension reaction a "universal" 15-mer M13 dideoxy sequencing primer was added followed by enzymatic extension and ligation. A double stranded 15 restriction fragment (BamHl-Hind III) was cut out of the partly double stranded circular DNA and ligated into pBR322 cut with BamHI and Hind III.

The obtained ligation mixture was used to transform E. coli and transformants harbouring a plasmid pMT319 containing the 20 gene encoding B(l-29)-A(l-21) of human insulin was identified.

Genes encoding B(l-29)-Ala-Ala-Lys-A(l-21) and B(l-29)-Ser-Lys-A(l-21) were made accordingly by insertion of a fragment encoding MFal-B-C-A in the M-13 bacteriophage and site specific mutagenesis of the human proinsulin sequence with 25 chemically synthesized 30-mer and 27-mer deletion primers, respectively, and the above mentioned "universal" 15-mer M13 dideoxy sequencing primer. A double stranded restriction fragment (Xbal-EcoRl) was cut out of the partly double stranded circular DNA and ligated into pUC13 and pT5, respectively. By 30 transforwation arid retransfonnation of JR.. coli transformants harbouring a plasmid pMT598 containing the gene encoding B(l-29)-Ala-Ala-Lys-A(l-21) and pMT63G containing the gene encoding B(l-29)-Ser-Lys-A(l-21) were identified.

A gene encoding B(l-29)-Thr-Arg-Glu-Ala-Glu-Asp-Leu-35 Gln-Lys-A(l-21) was made in a similar way as described above by insertion of a fragment encoding MFal-B(l-29)-A(l-21) in a M13 mpll bacteriophage ant' site? specific mutagenesis of the B(l-29)-A(l-21) sequence with a chemically synthesized 46-mer deletion primer (5 • -CACACCCAAGAC'JAAAGAAGCI'CAAGACTTGCAAAGAGGCATTGTG-3 ' ) and the "universal" primer. Also, by a similar procedure a gene 5 encoding B(l-29)-Thr-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln--Lys-A(l-21) was constructed. 3. Plasmid constructions.

The gene encoding B(l-29)-A(l-21) of human insulin 10 (B'A) was isolated as a restriction fragment from pMT319 and combined with fragments coding for the TPI promoter(TPTp}(T.

Alber and G. Kawasaki. Nucleotide Sequence of the Triose Phosphate Isomerase Gene of Saccharomvces cerevisiae. J.Mol. Applied Genet. 1 (1982) 419 - 434), the MFal leader sequence (J. 15 Kurjan and I. Herskowitz,. Structure of a Yeast Pheromone Gene (KFa)i A Putative a-Factor Precursor Contains four Tandem Copies of Mature a-Factor. Cell 30 (1982) 933 - 94 3) and the transcription termination sequence from TPT of S.cerevisiae (TPIT). These fragments provide sequences to ensure a high rate 20 of transcription for the B'A encoding gene and also provide a presequence which can effect the localization of B'A into the secretory pathway and its eventual excretion into the growth medium. This expression unit for B'A (TPIp-MFal leader - B'A -TPIT was then placed on a plasmid vector containing the yeast 2\i 25 origin of replication am? a selectable marker, LEU 2, to give pMT344 , a yeast expression vector for B'A.

During in vivo maturation of a-factor in yeast, the last (C-terminal) six amino acids of the MFal leader peptide (Lys-Arg-Glu-Ala-Glu-Ala) are removed from the a-factor precursor 30 by the sequential action of an endopeptidase recognizing the Lys-7\rg sequence and an aminodipeptidase which removes the Glu-Ala residues (Julius, D. et al. Cell 32^ (1983) 839 - 852). To eliminate the need for the yeast aminodipeptidase, the sequence coding for the C-terminal Glu-Ala-Glu-Ala of the MFal leader was reiupve£ via in vitro mutagenesis. The resulting yeast, expression j>lasroid, pKT475, contains the insert coding for TPIp-MFal leader (minus Glu-Ala-Glu-Ala) - B'A - TPIT.

Tii a preferred construction the modified expression 5 unit was transferred to a stable, high copy number yeast plasmid CPOT, (ATCC No. 39685), which can l>e selected merely by the presence of glucose in the growth medium. The resulting yeast expression vector for B'A was numbered pMT479.

The fragment encoding MFal leader (minus Glu-Ala-Glu-10 Ala)-B(l-29)-Ala-Ala-Lys-A(l-21) was isolated as a restriction fragment from pMT598 and combined with fragments coding for the TPI promoter and the TPT terminator and transferred to the above mentioned high copy number yeast piasrnid CPOT. The resulting yeast expression vector for B(1-29)-Ala-Ala-Lys-A(1-21) was 15 numbered pMT610.

The fragment containing the insert TPIp- MFal leader (minus Glu-Ala-Glu-Ala)-B(1-29)-Ser-Lys-A(1-21)-TPIT was isolated as a restriction fragment from pMT630 and transferred into CPOT. The resulting yeast expression vector for B(l-29)-Ser-Lys-A(l-21) 20 was numbered pMT639.

The fragment containing the insert TPTp- MFal leader-(minus Glu-Ala-Glu-Ala) -B(l-29) -Thr-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Lys-A(l-21)-TPIt was inserted into a high copy number yeast plasmid DPOT, being a CPOT derivative containing a Sphl-BamHT-25 fragment of pBR322 inserted into a SpHl-BamHI fragment of CPOT. The resulting yeast expression vector for B(l-29)-Thr-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Lys-A(l-21) was numbered pll26. 4. Transformation Plasmids pMT344 and pMT4 75 were transformed into S. 30 cerevisiae lex» 2 mutants by selection for leucin prototrophy as described by Hinnen et al^.(A. Hinnen, J.B. Hicks and G.R. Fink. Transformation of Yeast. Proc.Nat.Aca.Sci. 75 (1978) 1929).

Plasmids pMT479, pKT610, pMT639 and pll26 were transformed into S. cerevisiae strains carrying deletions in the 35 TPI gene by selecting for growth on glucose. Such strains are normally unable to grow on glucose as the sole carbon source anc' grows very slowly on galactose lactate medium. This defect is due to a mutation in the triose phosphate isomerase gene, obtained by deletion and replacement of a major part of this gene with the S. cerevisiae LEU 2 gene. Because of the growth deficiencies there 5 is a strong selection for a plasmid which contains a gene coding for TPT. pMT479 contains the Schizo. pombe TPI gene.

. Expression of human insu1in precursors in_yeast Expression products of human insulin type were measured by radioimmunoassay for insulin as described by Heding, L. 10 (Diabetologia 8, 260 - 66, 1972) with the only exception that the insulin precursor standard in question was used instead of an insulin standard. The purity of the standards were about 98% as determined by HPLC and the actual concentration of peptide in the standard was determined by amino acid analysis. The expression 15 levels of immunoreactive human insulin precursors in the transformed yeast strains are summarized in Table 1.

Table 1 Expression levels of immunoreactive human insulin precursors in yeast.

Intnunoreactive insulin precursor Yeast strain Plasmid Construct (nmol/1 supernatant) MT 350 (DSM 2957) pMT 344 B(l-29)-A(l-21) 100 MT 371 (DSM 2958) pMT 475 B(l-29)-A(l-21) 192 MT 519 (DJM 2959) pMT 479 B(1-29)-A (1-21) 2900 KT 620 (DSM 3196) pMT 610 B(l-29) -Ala-Ala-Lys-A( 1-21) 1200 - 1600 MT 649 (DSM 3197) pMT 639 B(l-29)-Ser-Lys-A(l-21) 1600 ZA 426 pll26 B (1-29) -Thr-Arg-G lu-Ala-Glu- Asp-Leu-Gln-Lys-A( 1-21) 200 The isolation am" characterization of expression products are given in Examples 7-9 and 12 - 13. 6. Conversion of human insulin precursor into B30 esters of human insulin The conversion of the human insulin precursors into human insulin esters can be followed quantitatively by HPLC (high 5 pressure liquid chromatography) on reverse phase. A 4 x 300 mm "nBondapak CIS column" (Waters Ass.) was used and the elution was performed with a buffer comprising 0.2 M ammonium sulphate (adjusted to a pH value of 3.5 with sulphuric acid) and containing 26 - 50% acetonitrile. The optimal acetonitrile concentration 10 depends on which ester one desires to separate from the .insulin precursor. In case of human insulin methyl ester separation is achieved in about 26% (v/'v) of acet.oni tri lo.

Before the application on the KPLC column the proteins in the reaction mixture were preciio tated by addition of 10 15 volumes of acetone. The precipitate was isolated by centrifuga-tion, dried in vacuo, and dissolved in 1 M acetic acid.

EXPERIMENTAL. PART Example 1 Construction of a gene coding for B(1-29)-A(1-21)insulin Materials and Methods "Universal" 15-mer M13 dideoxy sequencing primer d(TCCCAGTCACGACGT), T4 DNA ligase and restriction enzymes were obtained from New England Biolabs. DNA polymerase I "Klenow fragment" and T. polynucleotide kinase were purchased frora P-L 32 Biochemicals. (t- P)-ATP (7500 Ci./mmol) was obtained from New England Nuclear. The support, for oligonucleotide synthesis was 2 '-O-dimethoxytratyl N -isobutyry1deoxyguanosme bound via a 3'-O-succinyl group to aminomethylatec"! 1% cross! inked polystyrene beads from Bachem.

Construct.jon of M13 mplO insHXAPst phage; The M13 mplO derived phage mplO insHX was constructed by cloning of the 284 bp large proinsulin coding Hind TJI-XbaT fragment, isolated from p285, into Hind III-XbaI cut M13 mplO 5 RF.M13 mplO RF is available from P-L Biochemicals, Inc.

Milwaukee, Wis. (Catalogue No. 1541).

M13 mplO insHXAPst was constructed from mplO insHX,RF by complete Pst.7 digestion followed by ligation and transformation of E. coli JM103. The resulting phage harbours the human 10 proinsulin coding sequences, with a 75 bp in frame deletion in the C-peptide coding region. Single stranded phage was prepared as described (Messing, J. and Vieira, J. (1982) Gene 19, 269 -276) .

Oligodeoxyiibonucleotide synthesis 15 The 19-mer deletion primer d(CACACCCAAGGGCATTGTG) was synthesized by the triester method on a 1% crosslinked polystyrene support (Ito, H., Ike, Y., Ikuta, S., and Ttakura, K. (1982) Nucl.Acids Res. 10, 1755 - 1769). The polymer was packed in a short column, and solvents and reagents were delivered 20 semi-automatically by means of en KPIrC pump and a control module. The oligonucleotide was purified af'ter deprotection by KPLC on a LiChrosorb RP18 column (Chrompack (Fritz, H.-J., Belagaje, R., Brown, F..L., Fritz, R.H., Jones, R.A., Lees, R.G., and Xhorana, H.G. (1978) Biochemistry 17, 1257 - 1267). 32 5'- P-labelling of oligodeoxyribonucleotide The 19-mer was labelled at the 5'end in e 60pl reaction mixture containing 50 mM Tris-FCl at pH 9.5, 10 mM MgCl_, 5 mM "^2 PTT, 0.4% glycerol, 120 pmole ATP, 50 jiCi of (y~~ P)-ATP (10 pmole), 120 pmole of oligonucleotide and 30 units of T4 polynu- cleotide kinase. The reaction was carried out at 37°C for 30 min., and terminated by heating at 100°C for 3 min. The labelled 32 oligonucleotide was separated from unreacted (7- "P)-ATP by chromatography on a column (1x8 cm) of Sephadex G50 superfine in 0.05 M triethylammonium bicarbonate at pH 7.5.

For colony hybridization the oligonucleotide was labelled without the addition of "cold" ATP as described (Boel, E., Vuust, J., Norris, F., Norris, K., Wind, A., Rehfeld, J., and Marcker, K. (1983) Proc.Natl.Acad.Sci. USA 80, 2866 - 2869).

Oligodeoxyribonucleotide primed DNA synthesis Single stranded M13 mplO insHXAPst (0.4 pmole was 32 incubcitec with the 19-mer 5'-( P)-labelled oligodeoxyiibonu-cl.eotide primer (10 pmole) in 20 pi of 50 mM NaCl, 20 mM Tris-HCl I>F 7.5, 10 mM MgCl2 and 1 mM DDT for 5 min. at 55°C and annealed 10 for 30 min. at 11°C. Then 9 pi of d-NTP-niix consisting of 2.2 mM of each dATP, dCTP, dGTP, dTTP, 20 mM Tris-HCl, pH 7.5, 10 mM MgClj, 50 mM NaCl, 1 mM DDT was added followed by 7 units of E. coli DNA polymerase I (Klenow) . The mixture was kept for 30 min. at 11°C and heated for 10 min. at 65°C. 15-mer universal primer 15 for dideoxy sequencing (4 pmole) was added and the mixture heated at 65°C for an additional minute. After cooling to 11 °C 26 jil of solution containing 20 mM Tris-HCl pH 7.5, 10 mM MgClj# 10 mM DTT, 0.8 mM of each dATP, dCTP, dGTP, dTTP, 2.4 mM ATP and 103 units of T4 ligase was added followed by 9.5 units of E_. coli DNA 20 polymerase T (Klenow) . The final volume of the mixture was 64 jil. After incubation for 3 hours et 11CC 20 nl 4H sodium acetate was added, and the volume adjusted to 200 |il with TE-buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA).

The mixture was extracted twice with phenol/chloroform. 25 0.9 \tq (0.3 pmole) of the purified large fragment of pBR322 cleaved with BamHI and Hind TIT was added as carrier DKA. After ether extraction of the aqueous phase, the DNA was isolated by et.hai.ol precipitation.

Endonuclease digestion 30 The DNA, prepared, as described above, was digested respectively with 16 arid 20 units of restriction endonucleases BamHI and Hind III in a total volume of 22j.tl of buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DDT, 4 mM spermidine). The mixture was extracted with phenol/chloroform followed by ether and t.ho DMA v/as isolated by ethanol precipitation am" then dissolved in 12 jil Ho0. 2yl was used for electro- 4- phoresis on a 71* lire* 6% polyacrylamide gel.

Ligation To a part of the DNA (5 pi) was added a new poition of the purified large fragment of pBR322 cut with BamHI and Hind III (0.38 jig) and 400 units of T4 DNA ligase, in a total volume of 41 pi containing 66 ml-1 Tris-HCl, pH 7.4, 10 mM MgCl^, 1 mM ATP, 10 mM DDT, 4 0 po/ru] gelatine. Ligation was performed at 16°C for 16 10 hours.

Transformation .5 nl of the ligation mixture was used to transform CaClj treated E. coli MC 1000 (r , m+). The bacteria were plated on I.B-agar plates and selected for resistance to ampicillin (100 3 ng/ml). 2.6 x 10 colonies per pmole of M13 mplO insHX^JPst were obtained.

Colony hybridisation 123 transformed colonies were picked onto fresh ampicillin plates and grown overnight at 37°C. Colonies were trans-20 ferred to Whatman 540 filter paper and fixed (Gergen, J.P., Stern, R.H., and Wensink, P.C. (1979), Nucl.Acids Res. 7, 2115 -2136). A prehybridization was performed in a sealed plastic bac with 6 ml of 0.9 M NaCl, 0.09 M Tris-HCl pH 7.5 0.006 M EDTA, 0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum 25 albumin, 0.1% SDS and 50 jig/ml salmon sperm DNA for 2 hours at 65°C. Then 8.5 x 10 cpm of P-labelled 19-mer was added and hybridisation performed at 45°C overnight. The filter was washed with 0.9 K NaCl, 0.09 M sodium citrate three times at G°C for 5 min. and was then autoradiographed and washed once at 4 5°C for 1 30 ni n. ard autoradiographed again. After washing at 45°C, identification of 3 colonies containing mutated plasmid was possible.

Endonuclease analysis of mutated plasmids Plasmids from the suppose*' riiUtani colonies were prepared by a rapid method (Ish-Horowicz, D. and Burke, J.F. (1981), Nucl.Acids Res. 9, 2989 - 2998), digested with a mixture 5 of BamHI and Hind III and then analysed by electrophoresis on a 2% agarose gel. The presence of a 179 bp fragment confirmed that the 3 colonics container? i>-utant plasmid.

Retransformation The colonies identified "mutant" contain plasmids 10 which are the progeny of a heteroduplex. Pure mutant could be obtained by retransformation of CaCln treatec? E. coli HC1000 — + (r , m ) with plasmid from 2 of the mutant, colonies. From each plate 5 ampicillin resistant clones were isolated, plasmid DNA was prepared and analysed by endonuclease cleavage as mentioned 15 above. 3 out of 5 and 5 out of 5 respectively were shown to be pure mutant. One plasmid pMT319 was selected for further use.

DNA sequence analysis ng of pMT319 was cleaved with BamHI under standard conditions, phencl extracted and ethanol precipitated. Filling in of the BamHI sticky ends was performed with Klenow DNA polymerase I, dCTP, dGTP, dTTP, and a-32P-dATP.

After phenol extraction and ethanol precipitation the 32 DNA was digested with EcoRJ. The -P labelled fragment with the deletion was purified by electrophoresis on a 2% agarose gel and 25 sequenced by the Maxam-Gilbert method ( I'axam, A. and Gilbert, W. (1980) Methods in Fnzymology 65, 499 - 560).

Example 2 Construction of a yeast plasmid_pMT344 for expression of B(l-29)-A(1-21) of human insulin (B'A).

Plasmid pMT319 containing the gene coding for B'A and constructed as explained above was cut with restriction enzymes Hi rid IT J and Xbal and a 0.18 kb fragment was isolated (T. Maniatis, E.F. Fritsch, and J. Saiubrook. Molecular Cloning. Cold Spring Harbor Press 3 982) from a 2% egerose gel. Similarly a fragment (6.5 kb Xhol - Kind TT7) containing the S. cerevisiae TPT promotor (TPJp) (T. Alber and G. Kawasaki. Nucleotide Sequence of the Triose Phosphate 7somerase Gene of Sarrharomvces 5 cerevisiae, J.Mol. Applied Genet. 1 (1982) 419 - 434) and the MFal leader sequence (J. Kurjan and T. Kerskowitz, Structure of a Yeast Pheromone Germ (KFa): A Putative a-Factor Precursor Contains four Tandem Coi>ies of Mature a-Factor. Cell 30 (1982) 933 - 94 3) was isolated from plasmid p285 constructed as 10 described in US-patent application S.N. 547,748 of November 1, 1983. P285 contains the insert TPIp-NFal leader -B-C-A- TPIT and was deposited 5n yeast strain Z33 (ATCC No. 20681). A fragment (0.7 kb Xbal - BamHI) containing the TPJ transcription termination sequences (TP7^) (t. Alber and G. Kawasaki, 15 Nucleotide Sequence of the Triose Phosphate 7somerase Gene of Sacchoromyces cerevi siae. CT.tfol . Applied Genet. 1 (1982) 419 -434) was also isolated from p285. Finally a 5.4 kb Xhol - BamHI fragment was isolated from the yeast, vector YFpl3 (J.R. Broach. Construction of High Copy Yeast Vectors Using 2pm Circle 20 Sequences. Methods Enzymology 101 (1983) 307 - 325). The above foui fragments were ligated (T. Maniatis, E.F. Fritsch, and J. Sambrook. Molecular Cloning. Cold Spring Harbor Press 1982) and transformed into E. coli (T. tfanietis, E.F. Fritsch, and J. Sambrook. Molecular Cloning. Cold Spring Harbor Press 1982) 25 selecting for ampicillin resistance. Plasmids were isolated from the transformants and the structure of one of these, pMT344, verified by restriction mapping. The construction and main features of pf*T244 are outlined in fig. 1.

Example 3 Construction of a yeast, plasmid pMT475 for exj>ression of B (1 -29)-A(1-21) of human insulin (B'A) after a modified MFal leader.

To construct a plasmid for the expression of B'A after a MFal leader (J. Kurjan and I. Herskowitz, Structure of a Yeast. Pheromone Gene (MFa): A Putative a-Factor Precursor Contains four 35 Tandem Copies of Mature a-Factor. Cell 30 (1982) 933 - 943) lacking its last four amino acids (Glu-Ala-Glu-Ala), the 0.14 kb Xbal - EcoHTJ f ragn^nt cont.ai ni ng the A and part of the B' sequences was isolated from pMT319. Likewise the 5' proximal part of the gene was isolated as a 0.36 kb EcoRI - EcoRIT fragment 5 from pM215. Plasmid pM215 was constructed by subclone ng the EcoRI - XbaT fragment, containing the proinsulin B-C-A gene froir p285 into pUC13 (constructed as described for pUC8 and pUC9 by Vieira et al., Gene 19: 259 - 268 (1982)) and subsequent in vitro loop-out removal of the 12 bases coding for Glu-Ala-Glu-Ala at the 3 0 junction between FFal leader and proinsulin B-C-A gene. These two pieces covering the B'A gene were ligated to EcoRI - Xbal digested pUC1.3 vector (sec fig. 2) to give pMT473. The modified gene contained within a 0.5 kb EcoRI - XbaT fragment was isolated from pMT473 and then ligated to two fragments (4.3 kb Xbal -15 EcoRV and 3.3 kb EcoRV - EcoRI) from pMT342. pMT342 is the yeast vector pMT212 with an inserted TPIp-MFal leader - B-C-A - TPIT-The resulting plasmid, pMT475, contains the insert: TPIp - MFal leader (minus Glu-Ala-Glu-Ala) - B'A - TPIT« The construction of plasmids pMT342, pMT473 and pMT475 is outlined in fig. 2. The 20 construction of the vector pl-o*212 is shown in fig. 3. Plasmid pMLB1034 is described by M.L. Berman et al., Advanced Bacterial Genetics, Cold Spring Harbor (1982), 49 - 51 and pUC12 was constructed as described for pUC13 (Vieira et al, ibid.).

Example 4 Insertion of the B_( 1-29)-A(1-21) (B'A? gene into a stable yeast plasmid pMT4 79.

The modified B'A gene from pMT4 75 was isolated as a 2.1 kb BamHT - partial Sphl fragment and ligated to an approximately 11 kb BamHI - Sphl fragment of plasmid CPOT (ATCC No. 39 685) to 30 give plasmid pfTT479 (fig. 4). Plasmid CPOT is based on the vector Cl/1 which has been modified by substituting the original pBR322 Bgll - BamHI fragment with the similar Bgll - BamHT fragment from pUC13 and subsequent insertion of the S.pombe TPI gene (POT) (US patent application S.N. 614,734 filed on May 25, 1984) as a BamHI ScO T fragment to give CPOT. Beggs et al.. Nature 275, 104 patent application 01C34 09A.

Cl/1 is derived from poPB 24 8, - 109 (1978) as described in FP Example 5 Transformation cerevisae strain MT118 (a, leu 2, ura 3, trp 3) v/as grown on YPD medium (Sherman et a]., Kethods in Yeast Genetics, Cold Spring Harbor laboratory, 1981) to an OD,-nr, of 2.1. 100 ml bU U of culture was harvest.ee by centrifugation, washed with 10 ml of 10 water, recentrifuged and resuspended in 10 ml of (1.2 H sorbitol, 25 mM Na2EDTA pH= 8.0, 6.7 mg/ml di thiotrei t.ol) . The suspension was incubated at 30°C for 15 minutes, centrifugec7 and the colls resuspended in 10 ml of (1.2 M sorbitol, 10 mM Na2EDTA, 0.1 M sodium citrate pH = 5.8, 2 ing Novozym® 234 enzyme). The 15 suspension was incubated at 30°C for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2 M sorbitol ax*d ir- 10 ml of CAS (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris (Tris = Tris(hydroxymethyl)-aminometan) pH = 7.5) and resuspended in 2 ml of CAS. For transformation 0.1 ml of CAS-resuspended 20 cells were mixed with approximately 1 ng of plasmid pMT344 and left at room temperature for 15 minutes. 1 ml of (20% polyethylenglycol 4000, 10 mM CaCl2, 10 mM Tris pH = 7.5) was added and the mixture left for further 30 minutes at room temperature. The mixture was centrifuged and the pellet 25 resuspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD, 6.7 mM CaCl2, 14 |jg/ml leucine) and incubated at 3 0°C for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2 M sorbitol. 6 ml of top agar (the SC medium of Sherman et al., (Methods in Yeast Genetics, Cold Spring Harbor 30 Laboratory, 1981) with .leucine omitted and containing 1.2 M sorbitol plus 2.5% agar) at 52°C was added and the suspension poured on top of plates containing the same agar-solidified, sorbitol containing medium. Transformant colonies were picked after 3 days at 30°C, reisolated and used to start liquid 'cultures. One such transformant MT350 (=MT 118/pMT344) was chosen for further characterization.

Plasmid pMT475 was transformed into S-cerevisih<» strain 5 MT 362 (a,leu2) by the same procedure as above, and the transformant MT371 (=MT362/pMT475) isolated.

Transformation of pMT479 into strain E2-7B X E11-3C (a/a, Atpi/^tpi, pep 4-3/pep 4-3; this strain will be referred to as KT501) was performed as above with the following modifica-10 tions: 1) prior to transformation strain MT501 was grown on YPGaL (1% Bacto yeast extract, 2% Bacto j>eptone, 2% galactose, 1% lactate) to an ODg00 of 0.6. 2) the SOS solution contained YPGaL instead of YPD. One transformant MT519 (=MT50l/pMT479) was chosen for further characterization.

The transformed microorganisms TIT 350, MT 371 and MT 519 were deposited by the applicant with Deutsche Sammlung von Iiikroorganismen (DSM), Griesebachstrasse 8, D-3400 Gottingen, on May 15, 1984 and accorded the reference numbers DSM 2957, DSM 2958, and DSM 2959, respectively.

Example 6 Expression of B(l-29)-A(1-21) insulin in yeast Strains MT350 (DSM 2957) and KT371 (DSM 2958) were grown in synthetic complete medium SC (Sherman et ajL., Methods in Yeast Genetics, Cold Spring Harbor Laboratory 1981) with leucine 25 omitted. For each strain, two 1 liter cultures in 2 liter baJTled flasks were shaken at 30°C until they readied GBgQQnro ^ They were then centrifuged and the supernatant removed for further analysis.

Strain MT519 (DSM 29 59) was grown similarly but on YPD medium (Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) and to an 0D-.nri of 15, centrifuged and oOunm the supernatant separated for analysis as above.

Example 7 Expression of B( 1-29)-A (1-21) insulin in yeast, strain MT350 (DSM 2957) Yeast strain MT350 (DSM 2957) was grown as previously described in example 6 and expression products front 1.100 ml of supernatant from this strain weio isolated as follows: g of LiChroprep® RP-18 (Merck, art. 9303) were washed 3 times with 50 mM NH^HCO^, 60% EtOH and thereafter packed in a 6 x 1 cm column. The column was equilibrated with 50 ml of 50 mK NH.HCO,. 55 ml of 96% EtOH were added to 1100 ml of the 4 3 yeast supernatant, and the mixture was applied to the column overnight (flow: 70 ml/h).

The column was washed with 10 ml of 0.5 M NaCl and 10 ml of H20, and the peptides were eluted with 50 mK of NH^HCO^, 60% EtOH. The eluate (5 ml) was concentrated by vacuum centri-fugation to 1.4 ml (to remove the ethanol), and the volume was adjusted to 10 ml with 25 mK of KEPF.S buffer pH = 7.4. The sample 15 was applied to an anti insulin inuuunoabsorpt i on column (AIS column) (2.5 x 4.5 cm) which had been washed 4 times with 5 ml of NaFAM-buffer (Heding, L., Diabetologia 8, 260-66, 1972) and twice with 5 ml of 25 mM HEPES-buffer prior to the application. After the application, the column was allowed to stand for 30 min. at 20 room temperature and was thereafter washed 10 times with 4 ml of 25 mK KEPES buffer. The peptides were eluted with 20% KAc. The pH value of the eluate was adjusted to 7.0 with NH^OH, and the pool was concentrated to 500 jil by vacuum rotation.

The sample from the previous step was further purified 25 on HPLC. on a 10n Waters jiBondopak C-18 column (3.9 x 300 mm). The A and B buffers were 0.1% TFA in H20 and 0.07% TFA in MeCN, respectively. The column was equilibrated with 25% B (flow: 1.5 ial/min.) end the peptides were elu.ted with a linear gradient of MeCN (1%/mi.n.) and detected at 276 nm. The yield in each step of 30 the purification was determined by radioimmunoassay as previously described, and Table 2 summarizes the purification. The overall yield was 68%.

Table 2 Purification of expression products from yeast strain HT350 35 supernatant Purification step Volume (ml) insulin (nmol) Supernatant 1100 110 RP-18 10 116 Anti-insulin Sepharose 0.5 116 HPLC 2.5 75 x) Dilution effect way oiiscive^ in this sample Only one peak containing immunoreactive B(l-29)-A(l-21) insulin material was «?et t»ct.ed from the HPLC column. Peptide 10 material from this peak was isolated ant' subjected to amino acid sequence analysis. The sequence analysis was performed with a Gas Phase soquencci (Applied Riosystem Model 170A) as described by Hewick, R.M. et al. (J.Biol.Chem. 256, 7990-7997, 1981). From the sequencing results it could be concluded that the expression 15 products consisted of 3 peptides: (Glu-Ala)2~B(1-29)-A(1-21) insulin 89% Glu-Als-B(l-29)-A(1-21) insulin 2% B(1-29)-A(1-21) insulin 9% The peptides were present in the relative amount as indicated.

Example 8 Expression of B(l-29)-A(l-21) insulin in yeast strain MT371 (DSM 2958) Yeast strain MT371 (DSM 2958) Wias grown as previously 25 described in example 6 and expression products from 665 ml of supernatant from this strain were isolated as described in Example 7. The overall yield was 50 nmol, corresponding to 39%. Peptide material was isolated from the HPLC column and sequenced as described in Example 7. From the sequence results (18 residues 30 from the H-terminal) it could be concluded that the peptide was homogeneous B(l-29)-A(l-21) insulin.

Comparison of these results to the results obtained in Example 7 indicates the advisability of removing the Glu-Ala-Glu-Ala sequence from the C-terminal of the MF<*1 leader. It appears from Example 7 that the yeast dipeptidase enzyme does not function very efficiently in splitting off the Glu-Ala and Glu-Ala-Glu-Ala from the B(1-29)-A(1-21) insulin prior to secretion of the insulin precursor from the yeast cells.

Example 9 -Expression of B(l-29)-A(3-21) insulin in yoapt strain MT519 (DSM 2959) Yeast strain MT519 (DSM 2959) was grown as previously 5 described in example 6 and expression products from 70 ml of supernatant were isolated as described in example 7. The overall yield was 116 nmol, correspondsng to 57%. The peptide was sequenced as described in Example 7. As judged from the 42 residues identified from the N-terminal end, the peptide was 10 homogeneous B(l-29)-A(l-21) insulin. Approximately 5 rano] of peptide was hydrolyzed in 100 nl 6N HCl for 24 h at 110°C. The hydrolysate was analyzed on a Beckman Model 121M amino acid analyser. The following amino acid composition was found: Table 3 Amino acid analysis of purified B(1-29)-A(1-21) insulin Amino acid Found Theory Amino acid Found Theory Asx* 2.97 3 Val 3.37 4 Thr 1.77 2 lie 1.65 2 Ser 2.45 3 Leu* .65 6 Glx* 6.68 7 Tyr 3.51 4 Pro 1.33 1 Phe* 2.73 3 Gly* 3.95 4 Lys* 0.95 1 Ala* 1.22 1 His* 1.84 2 Cys 0.5 4 .54 6 Arg* 1.13 1 *) amino acid used for normalization.

Example 10 Construction of a yeast plasmid pMT610 for expression of B(1-29)-Ala-Ala-Lys-A(1-21) A 4.3 kb EcoRV-Xbal and a 3.3 kb F.coRT-EcoRV fragment 30 from pMT342 (see example 3) were ligated to a 0.6 kb EcoRI-Xbal fragment of pM215 (see example 3). The obtained plasmid pf*T462 harbours the insert MFal leader (minus Glu-Ala-Glu-Ala)-B-C-A. For converting the B-C-A encoding fragment into a B(l-29)-Ala- Ala-Lys-A(l-21) encoding fragment the modified site specific -mutagenesis procedure (K. Norris et al., ibid.) was used. A 0.6 kb EcoRI-Xbal fragment from pMT462 encoding MFal leader (minus Glu-Ala-Glu-Ala)-B-C-A was inserted into M13 mplO RF phage cut 5 with Xbal-FcoRT. Single strand M13 phage containing the above EcoRI-Xbal insert was incubated with a 30mer d (TTCACAATGCCCTTAGCGGCCTTGGGTGTG) primer (KFN15) and the "universal" 15-mer Ml 3 primer d(TCCCAGTCACGACGT) (see example 1), heated to 90°C for 5 minutes and slowly cooled to room 10 temperature in order to allow annealing. Then partly double stranded DNA was made by addition of a d-NTP-roix, Klenow Polymerase and T4 ligase.After phenol extraction, ethanol piecipitation and resuspension, the DNA was cut with restriction enzymes Apal,Xbal and EcoRI. After another phenol extraction, 15 ethanol precipitation and resuspension, the DNA was ligated to EcoRI-Xbal cut pUC13. The ligation mixture was transformed into an F.coli (r m ) strain and plasmids were prepared from a number of transforric-nts. Plasmid preparations were cut with FcoRl and Xbal and those preparations showing banc?s at both 0.5 and 0.6 kb 20 were retransformed into E .coli . From the retransformation a transformant harbouring only pUCl.3 with a 0.5 kb insert was selected. The sequence of the EcoRI-Xbal insert of this plasmid, pKT598, was then confirmed by the Maxam-Gilbert method to encode MFal leader (minus Glu-Ala-Glu-Ala)-B(l-29 )-Ala-Ala-Lys-A (1-21) . 25 The Xbal-EcoRI insert from pMT598 was provided with TPI promotor and TPI terminator by ligation of a 0.5 kb Xbal-F.coRT fragment of pKT598 with a 5.5 kb Xbal-F.coRI fragment of pT5. The construction of p75 harbouring the insert TPIp-MFal leader-B-C-A-TPIT is j llustrat.ec in fig. 8. The resulting plasmid pMT 601 containing 30 the insert TPIp-MFal leader (minus Glu-Ala-Glu-Ala)-B(1-29)-Ala-Ala-Lys-A(l-21) -TPI^ was cut with BamHI and partially with Sphl and the 2.1 kb fragment was inserted in CPOT cut with BamHI and Sphl. The resulting plasmid pMT610 was used for transformation of yeast.

Example 11 Construction of a yeast plasmid pI*T639 for expression of BJ1-29)-Ser-Lys-A(1-21) The BCA encoding fragment from pMT4 62 (see example 2 0) was converted into B(1-29)-Ser-Lys-A(1-21) by a procedure analogous with the procedure described in example 10 by site spocific mutagenesis with a mixture of a 27-mer 5 d(TCCACAATGCCCTTAGACTTGGGTGTG) primer KFN3 6 and the "universe]" 15-mer M13 primer. After filling in with Klenow polymerase and ligation with T4 ligase the partly double stranded DNA was digested with Apal, EcoRI and Xbal and ligated with the.5.5 kb Xbal - EcoRI fragment from plasmid pT5 (see example 10). After 10 transformation and retrarisf ormation into E.coli, a plasmid pMT 630 containing the insert MFal leader (minus Glu-Ala-Glu-Ala)-B(1-29)-Ser-Lys-A(1-21) was isolated and the sequence of the insert confirmed. The further procedure for obtaining plasmid pMT639 containing the insert TPIp-MFal (minus Glu-Ala-Glu-Ala)-15 B(l-29)-Ser-Lys-A(l-21)-TPI^ was as described in example 10. The construction of pKT639 is illustrated in Fig. 9.

Example 12 Expression of B(1-29)-Ala-Ala-Lys-A(1-21) in yeast, strain MT 620 S. cerevi siae strain 177501 (see example 5) was transformed with pMT 610 as described for pMT479 in example 5.

Transforntanl: colonies were picked after 3 days at 30°C, reisolated and used to start, liquid cultures. One such transformant MT 620 = (MT501/pMT610) was chosen for further characterization. MT620 was deposited by the applicant with Deutsche Sajnmlung von Mikroorgani smen (DSM), on January 16, 1985 and accorded the reference number DSM 319 6.

I-7T G20 was grown on YPD medium. A two liter culture in 2 liter baffled flask was shaken at 30°C to an 0D,ft/_ of 15. 600nm After ceritri fugation the supernatant was removed for further 30 analysis. The expression level determined by radioimmunoassay was 1.2 nmol/1. Expression products from 84 0 nil of supernatant, were purified as described in Example 7. (RP-18 column, Anti-insulin Sepharose and HPLC). The overall yield was 100 nmol corresponding to about 10%. Peptide material was isolated from the HPLC-column 35 and sequenced as described in Example 7. 35 Edman degradation cycles were carried out (Table 4). From the sequence results the position of the 3 cir.iino acid residue chains (Ala-Ala-Lys) Separating the B(l-29) and the A(1-21) chains was confirmed (see table 4).

Table 4 Sequence analysis of B(1-29)-Ala-Ala-Lys-A(l-21) isolated from the culture medium of strain MT 62 0.

?TH-amino acid Yield Cyclus No. " " "1 residue Phe (pmol) 3381 2 Val 1738 3 Asn 5169 4 Gin 2750 His 2045 6 Leu 1405 7 Cys - 8 Gly 1372 9 Ser 345 His 1105 11 Leu 2228 12 Val 1963 13 Glu 1219 14 Ala 1514 Leu 1793 "16 Tyr 1707 17 Leu 1354 18 Val 1765 19 Cys - Gly 882 21 Glu 1019 22 Arg 1100 23 Gly 1123 24 Phe 1492 Phe 2042 26 Tyr 1014 27 Thr 195 28 Pro 710 29 B qLys Ala 1173 1026 31" Ala 885 40 32 Lys 1175 33 A Gly 552 34 J le 518 Val 548 The average repetitive yield was 95.6%.

Example 13 Expression of B(1 -29)-Ser-Lys-A(1-21) in yeast strain J"T643 S. cerevisiae strain MT501 way transformed with pf"T639 as described for pMT479 in example 5.

One transformant MT643 = (MT501/pMT639) was chosen for further characterization. MT64 3 was deposited by the applicant at DSf' on January 1G, 1985 and accorded the reference No. DSM 3197.

KT643 was grown as described in example 12. After centrifugation the supernatant was removed foi further analysis. 10 The expression level of the insulin precursor determined by radioimmunoassay wes 1.6 j.imol/1 . Expression products from the supernatant from strain MT 64 3 was isolated as described in Example 7. The peptide Materia] isolated front the HPLC column was submitted to sequence analysis as described in 15 Example 7. From the sequence results (not shown) the position of the two amino acid residues chains (Ser-Lys) separating the B(l-29) and A(l-21) chains was confirmed.

Example 14 Conversion of B(1-29)-A(1-21) to Thr(But)-OBut(B30) human insulin 20 20 mg of B(l-29)-A(l-21) was dissolved in 0.1 ml of 10 M acetic acid. 0.26 ml of 1.54 M Thr(Bu^)-OBu*" in N,N-dimethylacetamide was added. The mixture was cooled to 12°C. 2.8 mg of trypsin dissolved in 0.03 5 ml of 0.05 M calcium acetate was added. After 72 hours at 12°C, the proteins were precipitated by 25 addition of 4 ni] of acetone, isolated by centrifugation and dried in vacuo. The conversion of B(1-29)-A(1-21) to Thr(Bi^)-OBu^(B30) human insulin was 64% by HPLC.

Example 15 Conversion of B(l-29)-A(l-21) to Thr-QMe(B30) human insulin 30 20 mg of B(1-29)-A(1-21) was dissolved in 0.1 ml of 10 ■ M acetic acid. 0.26 ml of 1.54 M Thr-OMe in a mixture of dimethyl sulphoxide and butane-1,4 diol 1/1 (v/v) was added. 1 mg of lysyl endopeptidase from Achromobacter lyticus (Wako Pure Chemical Industries, Osaka, Japan) in 0.07 ml of water was added. After 35 120 hours at 25°C, the proteins were precipitated by addition of 4 ml of acetone, isolated by centrifugation, ant' ('riec1 in vacuo. The conversion of B(l-29)-A(l-21) to Thr-OMe(B30) human insulin was 75% by HPLC.

Example 16 Conversion of B(l-29)-Ser Lys-A(l-21) to Thr-OBu*" (B30) human insulin _ _ mg of B(l-29)-Ser-Lys-A(1-21) was dissolved in 0.1 ml of a mixture of 34.3% acetic acid (v/v) and 42.2% N,N-dimet.hyl f ormami de (v/v) in water. 0.2 ml of 2 M Thr-OBu^ as 10 hydroacet.ate salt jn N,N-dimethy! formami de was added. The mixture was thermostated at 12°C. 2 mg of trypsin in 0.05 ml 0.05 K calcium acetate was added. After 24 hours at 12°C, the proteins were precipitated by addition of 4 ml of acetone, isolated by cent j i fiicati on and dried in vacuo. The conversion of B(l-29)-15 Ser-Lys-A(1-21) to Thr-OBu*" (B30) human insulin was 85% by FPLC.

Example 17 Conversion of B(l-29)-Ala-Ala-Lys-A(l-21) to Thr-OBu*"(B30) human insulin mg of B(l-29)-Ala-Ala-Lys-A(l-21) was dissolved in 20 0.1 ml. of a mixture of 34.3% acetic acid (v/v) and 42.2% N,N dimethylformamide (v/v) in water. 0.2 ml of 2 M Thr-OBu*^ as hydroacetate salt in N,N-dimethylforinamide was added. The mixture was thermostated at 12°C. 2 mg of trypsin in 0.05 ml 0.05 M calcium acetate was added. After 96 hours at 12°C, the proteins 25 were precipitated by addition of 4 "ml of acetone, isolated by centrifugation and dried in vacuo. The conversion of B(l-29)-Ala-Ala-Lys-A(l-21) to Thr-OBu*"(B30) human insulin was 84% by HPLC.

Example 18 Preparation of human insulin from various human insulin esters The human insulin esters in the crude acetone precipitates were purified by gelfi1tiation and anion exchange chromatography as described in Methods in Diabetes Research vol.1, p. 407 - 408 (Eds. J. Larner & S. Pohl (John VJiley Sons, 35 New York, 1984)). The method was applicable to any of the 3 human insulin esters. The cleavages of tli<r various ester groups, rendering human insulin in nearly 100* yields, were carried out t»y hydrolysis of Thr-OMe(B30) human insulin and by acidolysis with trifluoroacetic acid of Thr (Bu*") -OBu*" (B30) human insulin and 5 of Thr-0But(B3Q) human insulin as described ibid. p. 409.

Claims (2)

CLAIMS - 31 -

1. Human insulin precursors of the general formula B(1-29)-Xp-Y-A(1-21) 5 wherein Xn is a peptide chain with n naturally occurring amino acid residues, n = 0-33, Y is Lys or Arg, B(1-29) is a shortened B-chain of Bl 629 human insulin from Phe to Lys and A(1-21) is the A chain of human insulin, with the proviso that the peptide chain -X -Y- does not contain two adjacent, basic amino acid residues. 10

2. Human insulin precursors as claimed in Claim 1 substantially as described herein with reference to the Examples. 15 TOMKINS & CO. 20 25 30 35