CA1320162C - Method for stabilizing heterologous protein expression and vectors for use therein - Google Patents

Abstract

METHOD FOR STABILIZING HETEROLOGOUS PROTEIN
EXPRESSION AND VECTORS FOR USE THEREIN

Abstract of the Invention The present invention provides a method for stabilizing heterologous protein expression in bacteria by using a 3' truncated chloramphenicol acetyltransferase (CAT) gene fused in frame with a gene encoding a heterolo-gous protein. When expressed in a bacterial host, the resulting hybrid gene produces a fusion protein in re-coverable yield. Cleavage sites separating the CAT and heterologous protein are also provided to facilitate isolation and purification of the desired heterologous protein The invention further provides bacterial vectors containing the hybrid gene fusions for expression of the fusion protein comprising the desired heterologous protein.

Description

132~162 METHOD FOR STABILIZING HETEROLOGOVS PROTEIN
EXPRESSION AND VECTORS FOR VSE TH~REIN

Technical Field of the Invention The present invention relates generally to the field of biotechnology. More particularly, the invention relates to the fields of protein expression and recombi-nant DNA ~echnology to improve the yield of poorly ex-pressed mammalian polypeptides in bacterial hosts.

Backqround of the Invention Many eukaryotic proteins axe not capable of be-ing expressed in Escherichia coli in any measurable yield,or even if detectable, are not capable of being expressed at such commercially recoverable levels due to proteolysis of the foreign protein by the host. Small proteins (e.g., peptide~hormones of less than 100 amino acids) appear to be especially sensitive to degradation. The degree of proteolysis varies from host to host and protein to protein. Possibly the highest level of expression of a eukaryotic protein in E. coli has been observed with yamma interferon, which was expressed at approximately 60% of total cellular protein. The high lev~l of expression of a few eukaryotic proteins has been achieved because they reach a~concentration in the cell where they can aggregate into insolubIe masses called inclusion or refractile bvd-es (e.g., bovine growth honnone; Schoner et al (1985), ~ 3:151-154). In this~form, the eukaryotic protein is less susceptible to proteolysis.

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~ 32~162 Proteins which do not become insoluble on their o~n do in some cases form inclusion bodies if joined to another protein such as a procaryotic protein. A small number of prokaryotic proteins have been used in this man-ner: E. coli lacZ, ~E, and recA genes and the lambdacII gene, for example.
Chloramphenicol acetyltransferase (CAT) has been used as a selectable maxker (resistance to chloramphenicol), as an easily assayed enzyme to monitor the efficiency of both eukaryotic and prokaryotic expres-sion from different promoters (Delegeane, A.~., et al (lg87) Mol Cell Biol 7:3994-4002), regulatory sequences, and/or ribosome binding sites, and for gene fusions which join sequences encoding a eukaryotic protein to the nucleotide sequence encoding mature, native CAT (Buckley and Hayashi (1986) Mol Gen Genet 204:120-125; European Patent Publication 161,937, published 21 November 1985) or to the carboxy terminal fragment of CAT (usually retaining CAT activity)~
While the literature establishes that fusion proteins are useful to express heterologous proteins in bacteria and that the native CAT gene sequence has been used for such a purpose, efforts to use a truncated form of CAT to express or to increase the recoverable yield of heterologous, mammalian proteins such as amyloid protein A4-7Sl insert sequence, glucagon-like peptide I, adipsin/D, and lung surfactant SP-B and 5P-C, have not been reported. In light of the fact that many important protein~ cannot be successfully expressed in bacteria in any commercially recoverable yield, there is a need to ~ develop systems for the bacterial expression and recovery : of such proteins.

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Disclo~ure of the Invention One aspect of the invention concerns a method of stabilizing heterologous protein expression in a prokaryotic host comprising:
(a) constructing a hybrid gene comprising in sequential order, a 3' truncated chloramphenicol acetyltransferase (C~T) gene sequence fused in frame with a heterologous gene sequence encoding a mammalian polypeptide selec-ted from the group consisting of amyloid protein A4-751 insert sequence, glucagon-like peptide I, adipsin/D, lung surfac~ant protein SP-B and lung surfactant protein SP-C; wherein said polypeptide is normally not recoverable in bacterial expression systems, and wherein said hybrid gene, upon translation, produces a fusion protein in a recoverable yield;
(b) providing a vector for expression of said hybrid gene;
(c) culturing the prokaryotic host transformed with the expression vector; and (d) recovering the fusion protein.
A second aspect of the invention concerns a bacterial expression vector capable of enhancing the level of expression of non-stable, bacterially produced heterologous polypeptides comprising a hybrid gene having, in sequential order, a 3' CAT truncated gene sequence fused in frame to a heterologous gene sequence encoding a mammalian polypeptide selected from the group consisting of amyloid protein A4-751 insert sequence, glucagon-like peptide I, adipsin/D, lung surfactant protein SP-B and lung surfactant protein SP~C, wherein said polypeptide is normally not recoverable in bacterial expression systems;
whereby said truncated CAT gene sequence is capable of rendering the resulting fusion protein resistant to proteolytic degradation.
A preferred embodiment ~or both the method and vectox of the present invention employs a CAT coding . .

~4~ ~3?,~162 sequence of less than or equal to 180 amino acids, preferably between 73 and 180 amino acids. Although the resulting CAT protein is substantially reduced as compzred to the native CAT protein, surprisingly, it has been found that the truncated CAT protein substantially contributes to the stability of the expressed protein and therefore, permits recovery of an increased yield of the desired heterologous protein.
Yet another aspect of the invention provides an improved bacterial expression vector capable of Pnhancing the level of expression of non-stable, bacterially produced heterologous polypeptides wherein said vector contains a hybrid gene having in sequential order, a modified 3' truncated CAT gene sequence linked to a heterologous gene sequence. The improvement comprises altering one or more DNA codons of the truncated CAT gene to eliminate potential chemical cleavage sites within the CAT protein.
Other aspects of the invention will be readily apparent to those of skill in the art from the description and examples which follow.

Brief Description of the Drawinqs Figure 1 set3 forth the amino acid and cor-responding nucleotide sequences for a 241 amino acid (aa)CAT-hANP hybrid protein containing an endoproteinase Glu-C
proteolytic cleavage site. The amino terminal portion of this hybrid protein encodes the first 210 amino acids of CAT, which sequence is extensively referred to throughout the present invention.
Figure 2 illustrates a series of vectors and synthetic fragments used for cloning and expression of the CAT-hANF hybrid proteins of the invention Figure 2A
depicts an EcoRI-PstI synthetic fragment containing the E.
coli trp promoter-operator sequence, a ribosomal binding site, and downstxeam cloning sites. Figure 23 is a restriction site and function map of plasmid pTrp233.
Figure 2C is a restriction site and function map of plasmid pCAT21. Figure 2D is an EcoRI-HindIII synthetic fragment encoding the hANP (102-126) gene preceded by an endoproteinase Glu-C cleavage site. Figures 2E through G
are restriction site and function maps of plasmids phNF75, pChNF109, and pChNF121, respectively. Figure 2H depicts a synthetic 1-73 aa CAT gene sequence contained withi~ NdeI-HindIII fragment. Figure 2I is a restriction site and function map of plasmid pChNF142 wherein site-specific mutagenesis was used to substitute Tyr and Ser codons for residues 16 and 31, respectively, of the CAT gene.
Figure 3 illustrates two different preparative SDS-polyacrylamide gels. Figure 3A is an SDS-lS polyacrylamide gel of the CAT-A4-7Sli hybrid protein.
Lane 1 = molecular size standards; Lane 2 = induced W3110 (pCAPil32); Lane 3 - induced W3110 (pTrp83) vector control; Lane 4 = uninduced W3110 (pCAPil36); and Lane 5 =
induced W3110 (pCAPil36). Figure 3B is an SDS-polyacrylamide gel of the CAT-GLP-I hybrid protein. Lane 1 = molecular size standard; Lane 2 = uninduced W3110 (pCGLP139); Lane 3 = induced W3110 (pCGLP139); and Lane 4 = induced W3110 (pTrp83~ vector control.
Figure 4 illustrates the amino acid and cor-responding nucleotide sequences for a CAT-A4-751i hybrid protein and a CAT-GLP-I hybrid protein of the invention.
Figure 4A depicts the first 73 codons encoding the amino terminus of the CAT protein joined in-frame to the synthetic A4-751i gene preceded by a chemical cleavage and site encoded by Asn-Gly. Figure 4B depicts the first 73 codons encoding the amino terminus of the CAT protein joined in-frame to the synthetic GLP-l gene preceded by a Met codon.
Figure 5 illustrates two plasmids, pCAT73 and pCAT210,-in which the gene for tetracycline resistance is restored in these CAT expression vectors.

~ ~2~1 62 Figure 6 is the nucleotide sequence and cor-responding amino acid sequence of the SP-B expression construct pC210SP-B from the EcoRI site preceding the trP
promoter region through the HindIII site containing the translation stop codon. The CAT, linker, and SP-B regions are identified therein, respectively, by the arrows.
Figure 7 is a preparative SDS-polyacrylamide gel of the CAT:SP-B fusion protein. Lane A = molecular size standards; Lane B = induced W3110 cells containing pTrp233 vector control; and Lane C = induced pC210SP-B/W3110 cells.
Figure 8 illustrates the nucleotide sequence and corresponding amino acid sequence of the 251 residue CAT:SP-C fusion protein from plasmid pC210SP-C. The CAT
qene, linker sequence and SP-B gene are sequentially identified therein by the arrows.
Figure 9 provides the molecular wsight determinations for Pach of the CAT:SP-C fusion proteins.
Lane A = molecular size standards; Lane B = induced W3110 cells containing pTrp233 vector control; Lane C = induced pC106SP-C; Lane D = pC149SP-C; Lane E = pC179SP-C; and Lane F = pC210SP-C.
Figure 10 provides the cDNA and amino acid sequences for human adipsin/D.
Modes for CarrYing Out the Invention A. Definitions As used herein the term "stabilizing protein expression~ refers to a property of a fusion protein responsible for inhibiting proteolysis of a foreign protein by a recombinant host cell.
~ Insoluble~ as referred to proteins intends a condition wherein a protein may be recovered only by extraction with dstergents or chaotropic agents. Usually, .

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insoluble proteins are formed as a consequence of intracellular aggregation of the cloned gene products.
~ High protein expression" or "enhanced protein expxession~ refers to a level of expression wherein the fused protein can comprise 10% or more of the total protein produced by each cell. A preferred range for high protein expression levels is from 10-20~ of total cell protein.
As used herein, ~'non-recoverable" refers to a level of expression wherein the desired protein may be detected using sensitive techniques, e.g., Western blbt analysis, yet the protein is not commercially recoverable using conventional purification techniques such as SDS
polyacrylamide gel el~ctrophoresis, gel filtration, ion exchange chromatography, hydrophobic chromatography, af-finity chromatogrAphy, or isoelectric focusing.
IlMammalian'' refers to any mammalian species, and includes rabbits, mice, dogs, cats, primates and humans, preferably humans.
As used herein, the term "heterologous~ proteins refers to proteins which are foreign to the host cell transformed to produce them. Thus, the host cell does not generally produce such proteins on its own.

B. CAT Fusions CAT encodes a 219 amino acid mature protein andthe gene contains a number of convenient restriction endonuclease sites (5'-PvuII, EcoRI, DdeI, NcoI, and ScaI-3l) ~hroughout its length to test gene fusions for high level expression. These restriction sites may be used for ease of convenience in constructing the hybrid gene sequences of the invention or other sites within the gene sequence may ~e generated using t~chniques commonly known to those of skill in the art. Any of the resulting CAT
sequences are considered useful so long as the resulting .

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132~1 62 CAT fusion retains the ability to enhance the expression of the desired heterologous peptide.
The expression constructs of the invention can employ most of the CAT-encoding gene sequence or a substantially truncated portion of the sequence encoding an N-terminal portion of the CAT protein linked to the gene encoding the desired heterologous polypeptide. In one embodiment of the invention, the CAT portion of the fusion codes for about the N-terminal one-third of the CAT
sequence.
The expression constructs exemplified herein, which demonstrated enhanced levels of expression for a ~ variety of heterologous proteins, utilize a number of varying lengths of the CAT protein ranging in size from 73 to 210 amino acids. The 73 ~mino acid CAT fusion component is conveniently form~d by dige~ting the C~T
nucleotide sequence at the EcoRI restriction site.
Similarly, the 210 amino acid CAT fusion component is formed by digesting the CAT nucleotide sequence with ScaI.
These, as well as other CAT restriction fragmen~s, may then be ligated to any nucleotide sequence encoding a desired protein to enhance expression of the desired protein.
Significantly, although the expression level of fusion protein ~approximately 15-20~ of total cell protein) was similar for the CAT (106 amino acid) - SP-C
fusion and the CAT (210 amino acid) - SP-C fusion, it can be een that the former case actually represents a significant increase in expression level for the desired SP-C polypeptide, since the SP-C polypeptide constitutes a substantially larger proportion of the total fusion protein in the former case. The ability to increase expression level for the desired polypeptide by reducing the size of the fused CAT protein sequence was quite an unexpected finding in view of the experience of the prior art. In general, the prior art experience has been that . .

~32~62 reduction in size of the bacterial leader sequence does not result in increased production of the fused heterologous polypeptide due to a concomitant larger reduction in the expression level of the fusion protein.
With one exception, the various CAT-heterologous fusion proteins exemplified herein were found to be expressed in the range of approximately 10-20% of the total cell protein. Thus, the versatility of the CAT fu-sions, that is, the ability to use a variety of CAT coding sequences having the ability to enhance the expression of a desired protein, allows great flexibility of choice when construc~ing CAT hybrid genes.
The reading frame for translating ths nucleotide sequence into a protein begins with a portion of the amino terminus of CAT, the length of which varies, continuing in-frame with or without a linker sequence into the protein to be e~pressed, and terminating at the carboxy terminus of the protein. An enzyma~ic or chemical cleav-age site may be introduced downstream of the CAT sequence to permit recovery of the cleaved product from the hybrid protein. Such cleavage sequences are known in the art as are the conditions under which cleavage can be effected.
Following cleavage, the desired heterologous polypeptide can be recovered using known techniques of protein purification. Suitable cleavage sequences include, without limitation, cleavage following methionine residues (cyanogen bromide), glutamic acid residues (endoproteinase Glu-C), tryptophan residues ~N-chlorosuccinimide with urea or with sodium dodecyl sulfate (SDS)) and cleavage between asparagine and lysine residues (hydroxylamine).
To avoid internal cleavage within the CAT
sequence, amino acid substitutions can be made using conventional site specific mutagenesis techniques (Zollerr M.J., and Smith, M. (1982), Nuc Acids Res 10:6487-6500, and Adelman, J.P., et al (1983), DNA 2:183-193). This is conducted using a synthetic oligonucleotide primer com-.

1 ~'2~1 62 plementary to a single-stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Of course, these substitutions would only be performed when expression of CAT is not significantly affected. Where there is only one internal cysteine residue, as in the short CAT sequence, this residue may be replaced to help reduce multimerization through disulfide bridges.

C. CAT Fusion Vectors Procaryotic systems may be used to express the CAT fusion sequence; procaryotic hosts are, of course, the most convenient for cloning procedures. Procaryotes most frequently are represented by various strains of E. coli;
however, other microbial strains may also be used.
Plasmid vectors which contain replication sites, select-able markers and control sequences derived from a species compatible with the hos~ are used; for example, E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E coli species by Bolivar et al, Gene 2:95 (1977). pBR322 contains genes for ampicillin and tetracycline resistancer and thus provides multiple selectable markers which can be either retained or destroyed in constructi.ng the desired vector.
In addition to the modifications described above which would facilitate cleavage and purification of the product polypeptide, the gene conferring tetracycline resistance may be restored to the exemplified CAT fusion vectorq for an alternative method of plasmid selection and maintenance.
Although the E. coli tryptophan promoter-operator sequences have been exemplified in the present CAT vec~ors, different control sequences can be substituted for the ~ regulatory sequences and are considered to be within the scope of the invention. Com-monly used procaryotic control sequences which are defined 132~1~2 herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequence, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al, Nature 198:1056), the lambda-derived PL promoter (Shimatake et al, Nature 292:128 (1981)~ and N-gene rihosome binding site, and the trp-lac ~trc) promoter system (Amann and Brosius, Gene 40:183 (1985)).
Since the general utility of these CAT vectors have been established with very different mammalian peptides (ranging in protein size, the presence or absence of disulfide bonds, and being hydrophobic or hydrophilic in nature) vec~ors with unique restriction site~ may be created or substituted for the pBR322-derived vector il-lustrated in the examples.

D. Heteroloqous Protein Expression Amino terminal DNA sequences of CAT have beenfused to DNA sequences encoding human polypeptides for high lev~l expression in the bacterial host E. coli. The polypeptides described herein are relatively small mam-malian polypeptides ranging in size from about 30 to 76 amino acid residues. Atkempts to directly express, e.g., in a non-fused form, each of these polypeptides in bacteria have been unsuccessful, most likely due to the proteolytic degradation which occurs upon translation of the m~NA product. In the case of extremely hydrophobic polypeptides, even attempts to express such polypeptides using beta-galactosidase fusions produced detectable but very low level amounts of protein.
Examples of polypeptides that have been success-fully expressed to high level in bacteria using the truncated CAT fusions include a variety of mammalian polypeptides including amyloid protein A4-751 insert sequence, glucagon-like peptide I, adipsin/D, lung surfactant protein SP5 (SP-C), and lung surfactant SP18 ~.3~162 (SP-B). Preferably, the mammalian protein is of human origin, although other sources are also contemplated to be within the scope of this invention. A4-751 is a 57 amino acid sequence identified within the precursor for the A4 amyloid protein associated with Alzheimex's disease and shares homology with the Kunitz family of serine proteinase inhibitors (Ponte, P., et al (1988) Nature 331:525-527; Tanzi, R.E., et al (1988) Nature 331:528-530). Glucagon-like paptide I (GLP-I, 7-31) is a 31 amino acid hormone co-encoded in the glucagon gene which is a potent stimulator of insulin release (Mojsov, S., et al (1987) J Clin Inves 79:616-619j. Adipsin/D is a serine protease synthesized in and secreted from adipocytes (Zusalak, K.M., et al (1985) J Mol Cell Biol 5:419). Lung surfactant SP-B is a 76 amino acid hydrophobic protein.
Lung surfactant SP-C is a 35 amino acid hydrophobic protein. Both SP-B and SP-C greatly enhance spreading of surfactant phospholipids at an air:water interface.

E. Hosts Exemplified Host strains used in cloning and procaryotic expression herein are as follows:
For cloning an~ sequencing, and for expression of construction under control of mos~ bacterial pxomoters, E. coli strains such as MC1061, DHl, RRl, W3110, MM294, B, C600hfl, K803, HB101, JA221, and JM101 may be used.

F. General Methods ~ecombinant DNA methods are described in Maniatis et al (1982), Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, when not specifically cited in the following examples. Methods are also described in the literature for visualizing inclusion bodies, isolating them from cells, then solubilizing, purifying, and cleaving the hybrid protein (e.g., Itakura, X., et al ~1977) Science 19~.1056-1063; Shine, J., et al -13- ~ 3 2 a 162 (1980) Nature_285:455-461). Methods are also available, if necessary, for refolding ~he protein product (Creighton, T.E., Proceedinqs of Genex-UCLA Symposium, 1985, Kingstones (in press).

Examples I. Expression of Chloramphenicol AcetYltransferase-Human Atrial Natriuretic Peptide Hybrid Proteins in Escherichia coli.

A. Expression vector pChNF109.
Expression vector pChNF109 encodes a 241 amino acid CAT-hANP hybrid protein containing an endoproteinase Glu-C proteolytic cleavage site (Fig. 1). Most of the CAT
gene (amino acids 1-210) has been joined in-frame to the hANP(102-126) gene and cleavage site (26 amino acids) through a linker sequence (5 amino acids). The hANP
polypeptide comprises abou~ 10~ of the hybrid protein.
This vector was constructed from plasmids pTrp233, pCAT21, and phNF75 which supplied the plasmid backbone and trp promoter-operator, the CAT gene, and the h~NP(102-126) gene and cleavage site, respectively.
1. Construction of ~ChNF109.
Plasmid pTrp233 was constructed by insertion of a synthetic EcoRI-PstI fragment containing the E. coli trp promoter-operator sequence, a ribosomal binding site, and downstream cloning sites into plasmid pKK~33-2-NdeI which contains strong transcription termination signals, TlT2, `and the beta-lactamase gene. The synthetic fragment (see Fig. 2A) was assembled using the method of Vlasuk et al (1986), J. Biol Chem 261: 4789-4796 and its sequence confirmed by the method of Sanger et al (1977), Proc Natl Acad Sci USA 74:5463-5467 in M13mp8 and M13mp9. Plasmid ;~ :

:, .. .,: . -~23162 pKK233-2-NdeI (disclosed in U.S. patent 4,764,504 dated 16 August 1988) was digested with EcoRI and PstI, its termini dephosphorylated using calf intestinal phosphatase, and ligated with the synthetic EcoRI-PstI fragment. Plasmid pTrp233 was isolated (Fig. 2B) from E. Coli JA221 transformed to ampicillin resistance.
Plasmid pCAT21 was constructed by insertion of the CAT gene (from transposon Tn9, Alton and Vapnek, (1979) Nature 282:864-869) into plasmid pTrp233 under the control of the ~ promoter-operator. Plasmid pAL13ATCAT (a plasmid disclosed in Canadian application Serial No. 576,975 filed 8 September 1988), was digested with NdeI and HindIII and the approximately 750 bp NdeI-HindIII fragment containing the CAT
gene (with the initiating Met residue encoded at the NdeI
site) was purified using agarose gel electrophoresis. The CAT gene was ligated with NdeI and HindIII digested pTrp233 using T4 DNA ligase. From E. Coli MC1061 (Casadaban et al (1980), I Mo] Biol 138: 179-209) ampicillin-resistant .
transformants, plasmid pCAT21 was isolated (Fig. 2C).
Plasmid phNF75 was constructed by insertion of a synthetic hANP gene preceded by a proteolytic cleavage site into plasmid pBgal (Shine et al (1980), Nature 285:456).
Eight oligodeoxyribonucleotides (Fig. 2D) were assembled into a synthetic hANP(102-126) gene preceded by an endoproteinase Glu-C cleavage site (method of Vlasuk et al (1986), supra).
The synthetic DNA fragment (with a 5' EcoRI tail and a 3' blunt end) was ligated with EcoRI and SmaI restriction endonuclease digested M13mpl9 using T4 DNA ligase for the purpose of DNA sequencing (method of Sanger e~ al (1977), ~ ) A clone with the correct sequence, M13-hNF7, was digested with BamHI and BglII, the fragment containing the hANP gene purified by agarose gel electrophoresis, and the fragment ligated with BamHI-~' -15- 132~16~

digested and bacterial alkaline phosphatase dephosphorylated pTrp233 using ~4 DNA ligase. A plasmid with the insert in the orientation which gives adjacent HindIII, BamHI and EcoRI sites at the 3' end of the hANP
gene, phNF73, was identified by the siæe of the fragments generated ~y digestion with HindIII and PvuII. Plasmid phNF73 was digested with EcoRI, the hANP gene purified using polyacrylamide gel electxophoresis, and the gene ligated with EcoRI-digested and bacterial alkaline phosphatase dephosphorylated plasmid pBgal. From E coli MC1061 ampicillin-resistant transformants, plasmid phNF75 ~Fig. 2E) was identified by the size of the DNA fragments generated by digestion with PstI and HindIII.
Expression vector pChNF109 was constructed by insertion of DNA fragments containing CAT, hANP and the proteolytic cleavage site, and a linker sequence into plasmid pTrp233. Plasmid phNF75 was digested with EcoRI
and HindIII, the approximately 80 bp EcoRI-HindIII frag-ment containing hANP was purified by polyacrylamide gel electrophoresis, and ligated with EcoRI- and HindIII-digested pTrp233 using T4 DMA ligase. From E. coli MC1061 ampicillin-resistant transformants, plasmid phNF87 was isolated and digested with BamHI and the fragments were dephosphorylated using bacterial alkaline phosphatase. A
BamHI cassette containing the trp promoter-operator, ribosomal binding site, and large amino terminal fragment of the CAT gene was generated by digesting pCAT21 with ScaI, attaching BamHI synthetic linkers (5'-CGGATCCG-3') to the blunt termini using T4 DNA ligase, digesting the ligation with BamHI and purification of the approximately 740 bp BamHI fragment by agarose gel electrophoresis. The BamHI cassette and plasmid phNF87 were ligated using T4 ligase and ampicillin-resistant transformants of E. coli MC161 obtained. Plasmid pChNF109 (Fig. 2F), with the BamHI cassette in the orientation such that ~he C~T gene is fused in-frame ~o the endoproteinase Glu-C cleavage ~3~0~&~, site followed by the hANP gene, was selected on the basis of DNA fragment size in an EcoRI digest of the plasmid.

2. Expression of C~T(1-210)-hANP(102-126) Hybrid Protein From Plasmid pC NF103.
Plasmid pChNF109 expresses a CAT-hANP(102-126) hybrid protein under the control of the E. coli ~
promoter-operator. The plasmid was used to transform E.
coli W3110 (ATCC Accession No. 27325) to ampicillin resistance and one colony was grown in culture overnight at 37C in complete M9 medium containing M9 salts, 2 mM
MgS04, 0.1 mM CaC12, 0.4% glucose, 0.5~ casamino acids, 40 ug/ml tryptophan, 2 ug/ml thiamine hydrochloride, and 100 ug/ml ampicillin sulfate. The overnight culture was diluted 100-fold into the same M9 medium described above (uninduced culture) and into M9 medium in which the tryptophan had been replaced by 25 ug/ml of 3 beta-indoleacrylic acid (induced culture).
Expression was assessed after shaking the cultures for 6 hr at 37C. The uninduced culture had reached a high cell density (stationary phase) and the induced culture was still at a low cell density (exponential phase). Phase-contrast microscopy revealed cells of normal morphology in the uninduced culture and elongated cells containing several refractile inclusion bodies in the induced culture. Total cell protein samples were prepared by boiling cell pellets in Laemmli buffer for 5 min and were analyzed by electrophoresis through a 12% SDS-polyacrylamide gel followed by staining of the protein with Coomassie Blue.

B. ExPression Vector pChNF121.
Expression vector pChNF121 encodes a 99 amino acid CAT-hANP hybrid protein containing an endoproteinase Glu-C proteolytic cleavage site (Fig. 4A). Approximately one-third of the CAT gene (amino acids 1-73) has been .

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fused to the hANP(102~126) gene and proteolytic cleavage site (26 amino acids) without an intervening linker. The hANP polypeptide comprises 25% of the hybrid protein.
This vector was constructed from plasmids pChNF109 and phNF87 which supplied the amino terminal fragment of the CAT gene and the hANP gene and proteolytic cleavage site, respectively.

1. Construction of pChNF121.
Plasmid phNF87 was digèsted with EcoRI, its termini dephosphorylated with bacterial alkaline phosphatase, and ligated with an approximately 320 bp EcoRI fragment containing the trp promoter-operator, ribosome binding site, and amino-~erminus of the CAT gene. -This EcoRI cassette was purified from an EcoRI digest of PChNF109 using agarose gel electrophoresis. Plasmid pChNFl21 (Fig. 2G) was isolated from the ampicillin-resistant transformants of E. coli MC1061. On the basis of the si~e of the DNA fragments from a PvuII digest of the plasmid, the CAT and hANP genes were inferred to be fused in-frame to produce a hybrid protein.

2. Expression of CAT(1-73)-hANP(102-126) Hybrid Protein From Plasmid pChNF121.
Plasmid pChNF121 expresses a CAT-hANP(102-126) hybrid protein under the control of the E. coli trp promoter-operator. The plasmid was used to transform E.
coli W3110 (prototroph, 'rrpR~) to ampicillin resistance and one colony was grown in culture overnight at 37C in complete M9 medium (see Section A.2.). The overnight culture was diluted 100-fold into complete M9 medium (uninduced culture) and into M9 medium with 25 ug/ml 3-be~a-indole-acrylic acid xeplacing the 40 ug/ml tryp-tophan (induced culture).
Expression was assessed after shaking ~he cultures for 6 hr at 37C. The uninduced culture had ,~ ~ ' ' , .

-18- ~ 3 2 ~1 g2 reached a high cell density whereas the induced culture reached about one-third this density. Phase contrast microscopy revealed cells of normal morphology in the uninduced culture and elongated cells with several refractile inclusion bodies in the induced culture. Total cell protein samples were prepared by boiling cell pellets in Laemmli buffer for 5 min. and were analyzed by electrophoresis through a 12% SDS-polyacrylamide gel fol-lowed by staining of the protein with Coomassie Blue.
C. Ex~ression Vector pChNF142.
~ xpression ~ector pChNF142 encodes a 99 amino acid CAT-hANP hybrid protein containing a unique Trp residue following amino acid xesidue 73 of the CAT
protein, as a si~e for chemical cleavage. Approximately one-third of the CAT gene ~amino ac,ids 1-73) has been fused to the hANP(102-126) gene and chemical cleavage site t26 amino acids). This amino terminal fragment of CAT has been modified to substitute a Tyr residue for Trp~16] and a Ser residue for Cys[31] to remove the additional chemical cleavage site and reduce the multimerization of the hybrid protein through disulfide bridges. A synthetic hANP gene preceded by sequence encoding a Trp residue has been a~sembled for this vector.
1. Construction of pChNF142.
Plasmid pTrp233 was digested with EcoRI, its termini filled in with E. coli DNA polymerase I, Klenow fragment, and ligated with T4 DNA ligase (to remove the EcoRI restriction endonuclease cleavage site). From ampicillin-resistant transformants of ~. coli MC1061, plasmid pTrp81 was isolated and shown to resist cleavage by EcoRI. Plasmid pTrp81 was digested with NdeI and HindIII, purified by agarose gel electrophoresis, and ligated with a synthstic CAT gene ~ragment using T4 DNA
ligase. The synthetic NdeI-HindIII CAT gene fragment .~

, ~ --19--15~2~162 (Fig. 2H) was assembled from three pairs of oligo-deoxyribonucleotides as previously described. From ampicillin-resistant transformants of E. coli MC1061, plasmid pCAT127 was isolated and shown to contain the synthetic CAT fragment by digestion with EcoRI and AvaI.
The plasmid was digested with BamHI and HindIII, the BamHI-HindIII fragment containing CAT was purified by agarose gel electrophoresis, sequenced by the method of Sanger et al ~1977), supra, and the correct DNA sequence confirmed.
Plasmid pC~T127 was digested with EcoRI and HindIII and ligated using T4 DNA ligase with a pair of annealed synthetic oligodeoxyribonucleotides encoding hANP(102-126) preceded by a Trp residue on an EcoRI-HindIII DNA fragment. Plasmid pChNF142 (Fig. 2I) wasisolated from ampicillin-resistant transformants of E. coli MC1061. Insertion of the hANP gene was confirmed by the size of the DNA fragments in a BamHI and HindIII
digest of the plasmid. The sequence of -the hANP gene was confirmed from an EcoRI-ScaI agarose gel purified fragment from pChNF142.

2. Expression of CAT(1~73), T~r~l6l Ser[311-hANP(102-126) pChNF142.
~he expression of a modified CAT-hANP(102-126) hybrid protein is conducted in substantial accordance with the teaching of the previous examples A.2 and B.2.

II. E~ression of Chloramphenicol Acetyltransferase--Am~loid A4 Protein Insert (A4-751i~ Hybrid Proteins in Escherichla coli.
In the following examples high level expression of the 57 amino acid insert within the amyloid A4-751 protein was achieved by fusing a synthetic A~-751i gene to DN~ sequences encoding amino terminal fra~ments of CAT
under the control of the E. coli tryptophan promoter--20- ~32a~ 62 operator on a pBR322-derived plasmid. The synthetic A4-751i gene encodes amino acids 289-345 from amyloid A4-751 protein (Ponte et al (1988), Nature 331:525-527) preceded by a chemical cleavage site, Asn-Gly. Hydroxylamine cleavage of the hybrid protein between these two residues will yield the insert protein with a Gly residue at its amino terminus.

A. Expression Vector pCAPi132.
Expression vector pCAPil32 encodes a 132 amino acid CAT-A4751i hybrid protein containing a hydroxylamine cleavage si~e (Fig. 4A). Approximately the amino terminal third of the CAT gene (amino acids 1-73~ ha~ been joined in-frame to the A4-751i gene and clea~age ~ite (59 amino acids). The A4-751i protein comprises about 43~ of the hybrid protein. This vector was constructed from plasmids pTrp233 and pChNF121 and the synthetic A4-751i gene and cleavage site.

1. Construction of PcApil32.
Plasmid pTrp233 was digested with EcoRI and HindIII, purified by agarose gel electrophoresis, and ligated with the synthetic gene ~ncoding the A4-751i protein and cleavage site using T4 DNA ligase. The gene had been assembled from six oligodeoxyribonucleotides using previously described techniques and its sequence (Fig. 4A) co~firmed. Plasmid pAPil31 was isolated from ampicillin-resistant transformants of E. coli MCl061.
Insertion of the synthetic gene was confirmed by the size of the DNA fragments from a PvuI and BamHI digest of plasmid mini-prep DNA.
Plasmid pAPil31 was digested with EcoRI to lineari~e the vector and its termini dephosphorylated using bacterial alkaline phosphatase. Plasmid pChNF121 was digested with EcoRI and the approximately 320 bp ~coRI
fragmen~ containing the trp promoter-operator, ribosome ~ 3~0~
binding site, and amino terminus of the CAT gene ~amino acids 1-73) was purified by agarose gel electrophoresis.
This EcoRI cassette was ligated with the pAPil31 plasmid using T4 DNA ligase and ampicillin-resistant transformants s of MC1061 were obtained. On the basis of ~NA fxagment size in a PvuII digest of mini-prep plasmid DNA, plasmid pCAPil32 was isolated with an in-frame fusion of CAT and A4-751i sequences.

2. Expression of CAT( 1-73)-A4-751i Hybrid Protein From Plasmid pCAPil32.
Plasmid pCAPil32 expresses a CAT--A4-751i hybrid protein under the control of the E. coli ~ promoter-operator. The plasmid was used to transform E. coli W3110 to ampicillin resistance and one colony was grown in culture overnight at 37C in complete M9 medium. The overnight culture was diluted 100~fold into complete M9 medium which contains 40 ug/ml tryptophan (uninduced culture) and into complete M9 medium containing 25 ug/ml 3-beta-indoleacrylic acid instead of tryptophan (induced culture).
Expression was assessed after shaking the cultures for 6 hr at 37C. The uninduced culture had reached a high cell density whereas the induced culture was at a lower cell density. Phase contrast microscopy revealed cells of normal morphology in the uninduced culture and cells with ~pre-inclusion bodies~ in the induced culture. As used herein, "pre-inclusion bodies"
are defined as less refractile bodies which appear to convert in time to the more refractile "inclusion hodies~
as the hybrid protein accumulates in the cells. Total cell protein samples were prepared by boiling cell pellets in Laemmli buffer for 5 min and then analyæed by electrophoresis through a 12% SDS-polyacrylamide gel fol-lowed by staining with Coomassie Blue (Fig. 3~). ThisCAT(1-73)-A4-751i hybrid protein migrates between the : :

- --22~ ~ 32~6~

lysozyme (14,300 MW) and beta-lactoglobulin (18,400 M~7) protein standards on this gel. Vsing a Kontes fiber optic scanner and Hewlett-Packard Integrator to scan the gel, the hybrid protein was estimated to comprise about 7% of the total cell protein. This is a moderate expression le~el of E. coli but A4-751i comprises almost half of the hybrid protein.
To confirm the presence of A4-751i in the hybrid protein, Western blot analysis was carried out on an unstained 12% SDS-polyacrylamide gel o~ these protein samples. Protein was blotted to nitrocellulose and incubated with anti-A4-751i serum (prepared against a 16 amino acid synthetic pep~ide containing amino acids 11-26 of the 57 amino acid insert protein). After incubation with 125I-protein A (Amersham) the blot was placed on X-ray film at -70C for several days. The synthetic peptide anti-serum detected the hybrid protein as well as several other E. coli proteins.

B. Expression vector pCAPil36.
Expression vector pCAPil36 encodes a 274 amino acid CAT-A4-751i hybrid protein containing a hydroxylamine cleavage site. Most of the CAT gene (amino acids 1-210) has been joined in-frame to the A4-751i gene and cleavage site (59 amino acids) through a linker sequence (5 amino acids). The A4-751i polypeptide comprises about 21% of the hybrid protein. This vector was constructed from plasmids pAPil31 and pChNF109.

1. Construction of pCAPil36.
Plasmid pAPil31 was digested with EcoRI to linearize the vector and its termini dephosphorylated using bacterial alkaline phosphatase. From a partial EcoRI digest of pChNF109 an approximately 740 bp EcoRI
fragment containing the trp promoter-operator, the CAT
gene (amino acids 1 210), and linker sequence was purified .

.

' -23~

by agarose gel electrophoresis. This EcoRI cassette and vector pAPil31 were ligated using T4 DNA ligase and ampicillin-resistant transformants of E. coli MC1061 were isolated. From the size of DNA fragments in plasmid mini-preps digested with BamHI, plasmid pCAPil36 was isolatedwith the CAT gene and the synthetic A4-751i gene in-frame.

2. Expression of CAT(1-210)-A4-751i Hybrid Protein From Plasmid pCAPil36.
Plasmid pCAPil36 expresses a CAT-A4-751i hybrid protein under the control of the E. coli trp promoter-operator. The plasmid was used to transform E. coli W3110 to ampicillin resistance and one colony was grown in culture overnight at 37C in complete Mg medium. The overnight culture was diluted 100-fold into the same M9 medium (uninduced culture) and into M9 complete medium containing 25 ug/ml 3-beta-indoleacrylic acid instead of tryptophan (induced culture).
Expression was assessed after shaking the cultures for 5 hr at 37C. Both the uninduced and inducqd cultures reached high cell densities. Phase contrast microscopy revealed cells of noxmal morphology in the uninduced cultures and cells containing inclusion bodies or pre-inclusion bodies (50:50) in the induced cultures.
Total cell protein samples were prepared by boiling cell pellets in Laemmli buffer for 5 min and were analyzed by electrophoresis through a 12% SDS~polyacrylamide gel fol-lowed by staining with Coomassie Blue (Fig. 3A). This CAT-A4-751i hybrid protein migrates between the alpha-ch~motrypsinogen (25,700 MW) and ovalbumin (43,000 MW)protein standards on this gel. Using a Kontes fiber optic scanner and Hewlett-Packar~ In~egrator to scan the gel, the hybrid protein was estimated to comprises about 15% of total cell protein. This is moderately high level expres-sion for E. coli.
(*) Trademark .
,~:''"
.~

- -24- ~ 2 To confirm the presence of A4-751i ln the hybrid protein, Western blot analysis was carried out on an unstained 12% SDS-polyacrylamide gel of these protein samples. Using the method described above ~section II.
A.2.), the synthetic peptide anti-serum detected the hybrid protein as well as se~eral other E. coli proteins.

ssion of Chloramphenicol Acetyltransferase--Glucaqon-L e PePtide I ~-37) Hybrid Protein in Escherichia coli.
In the following example, high level expression of the 31 amino acid GLP-I(7-37) was achie~ed by fusing a synthetic GLP-I gene to DNA sequences encoding an amino terminal fragment of CAT under the control of the E. coli tryptophan promoter-operator on a pBR322-derived plasmid.
The synthetic gene encodes amino acids 7-37 of GLP-l (Mojsov et al (1987), J. Clin Invest 79:616-619) preceded by a Met residue. Treatment with cyanogen bromide releases the insulinotropic peptide.
A. Expression Vector PcGLpl39.
Expression vector pCGLP139 encodes a 105 amino acid CAT-GLP-I hybrid protein containing a cyanogen bromide clea~age site (Fig. 4B). Approximately the amino terminal third of the CAT gene (amino acids 1-73) has been joined in-frame to the GLP-l gene and cleavage site (32 amino acids). The GLP-I peptide comprises about 30~ of the hybrid protein. This vector was constructed from plasmids pTrp233 and pChNF109 and the synthetic GLP-I gene and cleavage site.

1. Construction_of pCGLP139.
Plasmid pTrp233 was digested with EcoRI and HindIII, purified by agarose gel electrophoresis, and ligated with the synthetic gene using T4 DNA ligase. The gene had been assembled from four oligodeoxyribo-25- 1~2~2 nucleotides and its sequence (Fig. 4B) confirmed. From ampicillin-resistant transformants of E. coli MC1061, plasmid pGLP138 was isolated. Insertion of the synthetic gene was confirmed by the failure of plasmid mini-prep DNA
to be cut by PstI.
Plasmid pGLP138 was digested with EcoRI to linearize the vector, its termini dephosphorylated using bacterial alkaline phosphatase, and ligated with the EcoRI
cassette from plasmid pChNF109 using T4 DNA ligase Plasmid pChNF109 had been digested with EcoRI and the ap-proximately 320 bp EcoRI fragment containing the trp promoter-oper~tor, ribosome binding site, and an amino terminal fragment of the CAT gene purified by agarose gel electrophoresis. Plasmid pCGLP139 was isolated from ampicillin-resistant transformants of MC1061. On the basis of DNA ragment size in an AvaI and PvuII digest of plasmid mini-prep DNA, the fusion of CAT and GLP-I
sequences was confirmed to be in-frame.

2. Expression of_CAT(1-73)-GLP-I(7-37) Hybrid Protei.n From Plasmid pCGLP139.
Plasmid pCGLP139 expresses a CAT-GLP-I hybrid protein under the control of the E. coli trp promoter-operator. The plasmid was used to transform E. coli W3110 to ampicillin resistance and one colony was grown in culture overnight at 37C in complete M9 medium. The overnight culture was diluted 100-fold into complete M9 medium which contains 40 ugtml tryptophan (uninduced culture) and into complete M9 medium in which 25 ug/ml 3-beta-indoleacrylic acid has been substituted for the tryptophan tinduced culture).
Expression was assessed after shaking the cultures for 6 hr at 37C. The uninduced culture had reached a high cell density whereas the induced cul~ure was at a lower cell density. Phase contrast microscopy revealed cells of normal morphology in the uninduced -26- 132~162 culture and elongated cells with three or more refractile inclusion bodies in the induced culture. Total cell protein samples were prepared by boiling cell pellets in Laemmli buffer for 5 min and were analyzed by electrophoresis through a 12% SDS-polyacrylamide gel fol-lowed by staining with Coomassie Blue (Fig. 3B). This CAT(1-73~-GLP-I(7-37) hybrid protein migrates between the bovine trypsin inhibitor (6,200 MW) and lysozyme (14,300 MW) protein standards. Using a Kontes fiber optic scanner and Hewlett-Packard Integrator to scan the gel, the hybrid protein was estimated to comprise about 20~ of the total cell protein. (Considering the number of inclusion bodies observed per cell, all of the hybrid protein may not have been solubilized in the Laemmli buffer, and this estimate may be low.) This is high level expression for E. coli.
The molecular weight of the hybrid protein is as predicted for this gene fusion. Amino acid composition analysis of the purified hybrid protein or protein sequencing of the peptide after cyanogen bromide cleavage can be performed to confirm its expression.

IV. CAT Fusion With Human SP-B and SP-C.
The mature forms of both human SP-C and SP-B are expressed as fusions with portions of bacterial CAT. The surfactant peptides are ~oined to the carboxy terminus of the CAT sequences through a hydroxylamine-sensitive asparagine-glycine linkage. The CAT-surfactant fusions are expressed from the tryptophan promoter of the bacte-rial vector pTrp233.
A. Expression Vector pC210SP-B.
SP-B expression vector pC210SP-B encodes a fu-sion protein of 293 residues in which 210 amino acids of CAT are joined to the 76 amino acids of SP-B through a linker of 7 amino acids containing ~he hydroxylamlne-sensitive cleavage site. Cleavage of the fusion with -27- ~ 3~162 hydroxylamine releases a 77 amino acid SP-B product containing the 76 residue mature form of SP-B, plus an amino-terminal glycine residue.
To construct pC210SP-B, the short EcoRI-HindIII
segment containing ANF sequences was removed from pChNF109, and replaced by a portion of human SP-B cDNA #3 extending from the PstI site at nucleotide (nt) 643 (Fig.
6) to the SphI site at nt 804. The EcoRI site wa~ joined at the PstI site through two complementary oligonucleotides encoding the hydroxylamine sensitive cleavage site as well as the amino-terminal residues of mature SP-B (oligo ~2307: 5 ' -AAT TCA ACG GTT TCC CCA TTC
CTC TCC CCT ATT GCT GGC TCT GCA-3' and oligo #2308: 5'-GAC
CCA GCA ATA GGG GAG AGG AAT GGG GAA ACC GTT G-3'). The lS ~I site was joined to the HindIII site of PTrp233 through a second se~ of complementary nucleotides encoding the carboxy~terminal residues of ma~ure SP-B (oligo #3313:
5'-ACC TTA CCG GAG GAC GAG GCG GCA GAC CAG CTG GGG CAG CAT
G-3' and oligo #3314: 5'-CTG CCC CAG CTG GTC TGC CGC CTC
GTC CTC CGG TA-3').
The expression plasmid was used to trans~orm E.
c _ stain W3110 to ampicillin resistance. Rapidly grow-ing cultures of pC210SP-B/W3110 in M9 medium were made 25 ug/ml IAA (3-beta indoleacrylate, Sigma I-1625) to induce the Trp promoter. By 1 hr after induction, refractile cytoplasmic inclusion bodies were seen by phase contrast microscopy inside the still-growing cells. 5 hr after induction, the equivalent of 1 O.D.550 of cells were pelleted by centrifugation, then boiled for 5 min in SDS
sample buffer for electrophoresis in a 12~ SDS-polyacrylamide gel followed by staining with Coomassie Blue (Fig. 7). Lane A = molecular size standards; Lane B
= induced W3110 cells containing pTrp233 vector control;
and Lane C = induced pC210SP-BJW3110. The predicted molecular weight of the CAT:SP-B ~usion protein is 45,000 daltons. The hybrid CAT:SP-B protein was es~imated to - -28- ~3~62 comprise 15-20~ of the total cell protein in the induced cultures.

B. CAT Fusions with SP-C.
A series of vectors wer0 constructed encoding fusion proteins in which mature human SP-C was fused to the carboxy termini of different portions of CAT through a hydroxylamine-sensitive asparagine-glycine linkage.
~Iydroxylamine cleavage of the fusion protein produced by each cons~ruct releases a mature SP-C of 35 amino acids which lacks the amino-terminal phenylalanine residue seen in a portion of natural human SP-C.

1. pC210SP-C.
The amino acid sequence of the 251 residue fu-sion protein encoded plasmid pC210SP-C. The 210 amino acids of CAT are joined to 35 amino acids of mature SP-C
through a linker of 6 amino acids. The mature SP-C
portion of the total fusion protein comprises 14%.
In Fig. 8 is shown the nucleotide sequence of pC210$P-C, in which th~ EcoRI-HindIII fragment of pC210SP-B containing SP-B sequences has been replaced by a segment of human SP-C cDNA #18 extending from the A~LI site at nucleotide 123 to the AvaII site at nucleotide 161. The EcoRI site of the CAT vector was joined to the SP5 ApaLI
site through two complementary oligonucleotides encoding the hydroxylamine sensitive cleavage site as well as the amino-terminal residues of mature SP-C (oligo #2462: 5'-AAT TCA ACG GCA TTC CCT GCT GCC CAG-3' and oligo #2463:
5'-TGC ACT GGG CAG CAG GGA ATG CCG TTG-3'). The A~aII
site of SP-C was joined to the HindIII site of pC210SP-B
through a second set of complementary nucleo~ides encoding the carboxy-terminal residues of mature SP-C and a stop codon (oligo #2871: S'-AGC TTA GTG GAG ACC CAT GA& CAG GGC
3S TCG CAC AAT CAC C~C GAC ~AT GAG-3' and oliqo #2372: 5'-GTC

.

, '' ' ' . -29-~3~al62 CTC ATC GTC GTG GTG ATT GTG GGA GCC CTG CTC ATG GGT CTC
CAC TA-3').

2~ ~C179SP-C.
The amino acid sequence of the 217 residue fu-sion protein encoded by pC179SP-C is a slight modification of the sequence shown in Fig. 8. In pC179SP-C, the 179 amino acids of CAT are joined to 35 amino acids of mature SP-C through a linker of 3 amino acids (Glu, Phe, Asn).
SP-C portion of the total fusion protein comprises 16%.
To construct pC179SP-C, a portion of the CA~
sequence was removed from pC210SP-C. S~arting with pC210SP-C, a DNA fragment extending from the Ncol site at nt 603 (Fig. 8) to the EcoRI site at nt 728 was removed, and the NcoI and EcoRI cohesi~e ends were rejoined with two complementary oligonucleotides (oligo #3083s 5'~CAT
GGG CAA ATA T~A TAC GCA AG-3' and oligo #3084: 5'-AAT TCT
TGC GTA TAA TAT TTG CC-3'). In effect, 31 residues of CAT, and 3 residues of the linker polypeptide are missing in the new fusion protein encoded by vector pC179SP-C.

3. pC14gSP-C.
The amino acid sequence of the 187 residue fu-sion protein encoded by pC149S~-C is a slight modification of the sequence shown in Fig. 8. In plasmid pC149SP-C, the 149 amino acids of CAT are ~oined to 35 amino acids of mature SP~C through a linker of 3 amino acids (Glu, Phe, Asn). The SP-C portion of the total fusion protein comprises 18.7%.
To construct pC1495P-C, a portion of the CAT
segment of pC210SP-C extending from the DdeI site at nt 523 (Fig. 8) to the EcoRI site at nt 728 was removed and replaced by a~set of two complementary oligonucleotides (oligo #3082: 5' TCA GCC:AAT CCC:G-3' oliqo #3081: 5'-AAT
35~ TCG GGA TTG GC-3').

:: :

,. .

, .

_30_ ~3 233 1 ~2 4. pC106SP-C.
The amino acid sequence of the 144 residue fu-sion protein encoded by pC106SP-C is a slight modification of the sequence shown in Fig. 8. In plasmid pC106SP-C, the 106 amino acids of CAT are joined to 35 amino acids of mature SP-C through a linker of 3 amino acids (Glu, Phe, Asn). The SP-C portion of the total fusion protein comprises 24%.
pCl06SP-C was constructed by replacing the EcoRI
fragment of pC210SP-C (nt 302 to nt 728, Fig. 8) with two sets of complementary oligos which were annealed, then ligated together through a region of homology (oligo #3079: 5 '-AAT TCC GTA TGG CAA TGA AAG ACG GTG AGC TGG TGA
TAT GGG ATA GTG TTC ACC CTT GT-3' was annealed with oligo 15 #3085: 5'-ACA CTA TCC CAT ATC ACC P,GC TCA CCG TCT TTC ATT
GCC ATA CGG-3'; oligo #3080: 5 ' -TAC ACC GTT TTC CAT GAG
CAA ACT GAA ACG TTT TCA TCG CTC TGG G-3' was annealed with oligo #3078: 5~-AAT TCC CAG AGC GAT GAA AAC GTT TCA GTT
TGC TCA TGG AAA ACG GTG TAA CAA GG& TGA-3').

5. Expression From SP-C Vectors.
Each SP-C expression vectox was used to transform E. coli strain W3110 to ampicillin resistance.
Rapidly growing cultures of expression strains were induced as described above. By 1 hr after induction, refractile cytoplasmic inclusion bodies were seen by phase contrast microscopy inside the still-growing cells. 5 hr after induction, the equivalent of 1 O.DL550 of cell~ were pelleted by centrifugation, then boiled for 5 min in SDS
sample buffer for electrophoresis in a 12% SDS-polyacrylamide gel followed by staining with Coomassie Blue. The results are provided in Fig. g wherein Lane ~ =
molecular size standards, Lane B = induced W3110 cells containing pTrp233 vector control; Lane C = induced pC106SP-C; Lane D = pC149SP-C; Lane E = pC179SP-C; Lane F
= pC210SPWC. The hybrid CAT:SP-C protein produced by each . , . , ~ .

i ~J~16~
vector is estimated to comprise 15-20~ of the total cell protein in the induced cultures.

v. Improved CAT Vectors for Expression of ~ybrid Proteins in Escherichia Coli.
In the following examples, the basic CAT gene fusion vector has been improved in several ways: (1) unique cloning sites are created for insertion of the gene to be expressed, (2) the CAT gene is modified to optimize cleavage andtor purification of the peptides, and (3) the gene conferring resistance to tetracycline is restored to provide an alternatlve method for plasmid selection and maintenance.

A. Expression Vectors ~CAT73 and PCAT210.
Expression vector pCAT73 contains genes confer-ring resistance to both ampicillin and tetracycline, unique EcoRI and HindIII cloning sites for insertion of genes to be expressed, and the amino terminal fragment (1-73) of the CAT gene. The cleavage site, included with the inserted ~ene, may not be unique. This plasmid is constructed from plasmids pBR322, pTrp233, pCAT21, and oli~odeoxyribonucleotides. Expression vector pCAT210 dif-fers from pCAT73 in that it contains the larger amino terminal fragment (1-210) of the CAT gene from which the EcoRI site at the sequence encoding residues 72 and 73 (Glu-Phe) ha-c been removed. ~An alternative codon choice preserves the Glu and permits the use of unique EcoRI and HindIII cloning sites.) Other DNA fragments encoding the amino terminus of the CAT gene, smaller than 73 amino acids or between 73 and 210 amino acids may also be constructed by insertion of an EcoRI site at the desired fusion point.

132~6~
l. Construction of pCAT73.
Restoration of the gene for tetracycline resist-ance requires restoring the BamHI-HindIII-EcoRI fragment of pBR322 to the CAT expression vector. Since the unique cloning sites desired for this vector are EcoRI and HindIII, this must be done in a manner which removes these sites but retains resistance to tetracycline. Since insertion of DNA at the HindIlI site upstream of the cod-ing region often prevents gene expression, this site i5 removed by creating a point mutation at the HindIII site.
Plasmid pBR322, was digested with EcoRI and HindIII and the vector backbone gel purified. The backbone was ligated with synthetic EcoRI-HindII fragments, which are formed by annealing pairs of oligonucleotides using T4 DNA
lS ligase. The fragments contain the normal EcoRI-HindIII
sequence with the exception of point mutations (G or C) at the first adenine of the recognition sequence 5'-AAGCTT-3'. An intermediate plasmid was isolated from ampicillin-resistant and tetracycline-resistant E. coli MC1061 transformants whose plasmid mini-prep DNA was not digested by HindIII.
A BamHI-EcoRI fragment no longer containing a HindIII site was purified from agarose gel electrophoresis from a BamHI and EcoRI digest of plasmid pTetHl. The fragment was ligated using T4 DNA ligase with plasmid pTrp233 which was also digested with ~amHI and EcoRI and agarose gel purified. Transformed wi~h the ligation, colonies of E. coli MC1061 were selected for ampicillin and/or tetracycline resistance. Plasmid pTrpT233 was resistant to both antibiotics.
In an alternate embodiment, digestion of pTrpT233 with EcoRI, blunting of the termini with DNA
polymerase I, Klenow fragment, and ligation with T4 DNA
ligase will eliminate the EcoRI site (which does not affect resistance to tetracycline). Tetracycline-resistant plasmid pTrpT234 which has lost undesirable ~3201 6~
HlndIII and EcoRI sites is isolated from colonies of E.
coli MC1061 transformed with this ligation.
The CAT gene is obtained as an NdeI-HindIII
fragment purified by agarose gel electrophoresis of an NdeI-HindIII digest of pCAT21. Plasmid pTrpdeltaHind was digested wikh NdeI and HlndIII, purified by agarose gel electrophoresis, and ligated with the CAT gene using T4 DNA ligase. From ampicillin (or tetracycline) resistant transformants of E. coli MC10Sl digested with EcoRI and HindIII to verify incorporation of the CAT gene, plasmid pCAT73 (Fig. 5A) is isolated.

2. Construction of PCAT210.
The BamHI-HindIII fragment containing the trp promoter-operator, ribosome binding site, and CAT gene is purified by agarose gel electrophoresis from a BamHI and HindIII digest of plasmid pCAT21. Site specific mutagenesis is carried out on the fragment using M13 and mutagenic oligodeoxyribonucleotides to convert the GAA
codon for Glu to GAG (also to Glu) within the EcoRI site, 5'-GAATTC-3'. One such plasmid, M13-CATdR, is digested with ScaI to linearize the vector and ligated with an EcoRI linker (for the same reading frame as in pCAT73) using T4 DNA ligase. From the transfectants, M13-CATR1, is isolated and digested with NdeI and HindIII. The new CAT gene is purified by agarose gel electrophoresis and ligated using T4 DNA ligase with NdeI~HindIII-digested plasmid pTrpT234. Plasmid pCAT210 (Fig. SB) is isolated from ampicillin (or tetracycline) resistant transformants f E coli MC1061.

B. Expression Vectors pCAT73-T and ~CAT73-M.
Expression ~ectors pCAT73-T and pCAT73-M are examples in which the amino acid sequence of CAT has been altered using site specific mutagenesis techniques to facilitate purification of the product protein. In these , .

:~ 3 2 ~
cases, the Trp residue at position 16 may be substituted with Tyr and the Met residue at position 67 may be substituted by Ile or Leu to eliminate potential chemical cleavage sites within CAT. In addition, the Cys at posi-tion 31 may also be subs~ituted using a conservative aminoacid alteration, that is, substitution with an amino acid which does not adversely affect biological activity.
Preferred residues include alanine, serine, leucine, isoleucine and valine, most preferred is serine. These la~ter alterations are intended to reduce multimerization through disulfide bridges.

C. Expression of Modified CAT-GLP-1 Plasmid pTrpdeltaHind contains the restored TetR
gene from pTrp233 (although the HindIII site has been eliminated), the Trpl6 to Tyr, Cys3l to Ser, and Met67 to Leu substltutions in the CAT gene sequence, and the GLP 1 gene (taught in Example III) fused in-frame to the modified CAT gene -through a methione residue. The vector was used to transform several E. coli strains including ~3110, MC1061, DHl, MM294 and RR1.
E. coli RRl transformants were more stable and appeared to have better induction/repression control of the Trp promoter than any of the other hosts. An alternative construction for this vector includes reversing the TetR gene (to avoid the back-to-b~ck placement of the Tet~ and Trp promoters in the present construct) to alleviate the stability problems observed using bacterial hosts other than RRI transformants.
VI. Construction of pTrpCAT72:Adipsin/D.
The coding sequence for mature human adipsin/D
was fused to pCAT72 to produce a fusion protein suitable, for example, to generate antisera agains~ human adipsin/D.

,, ,~

:L~2~162 A. Construction of pTrpCAT7? Q3Sl Plasmid pCAT72 Q3Sl was cons~ructed to eliminate Asn residues at which secondary cleavages can occur during hydroxylamine release of peptides fused to CAT. The A~n residues at amino acid positions 26, 51 and 78 of CAT were changed to Gln residues. At the same time, the single Cys at position 31 was changed to Ser to decrease the amount of aggregation seen with many CAT fusion proteins.
The vector pCAT72 Q3Sl was constructed as fol-lows: Oligos CAT72-1 through 6 (below) were annealed and ligated into pUC-9 which had been cleaved with NdeI ~nd EcoRI. In this way, the mutated CAT72 was joined to the polylinker region of the pUC plasmid. CAT72 Q3Sl with the polylinker was then removed from pUC by cleavage with NdeI
and HindIII, and inserted into pTrp233 b~tween NdeI and HindIII to yield pTrpCAT72 Q3Sl.

TATGGAGAAA AAAATCACTG GATATACCAC CGTTGATATA TCCCAATGGC
~60 70 ATCGTAAAGA ACATTTTGAG GCATTTCA

CAAAATGTTC TTTACGATGC CATTGGGATA TATCAACGGT GGTATATCCA
TGATTTTTT TCTCCA

TCAGTTGCT CAATCTACCT ATCAGCAGAC CGTTCAGCTG GATATTACGG

CCTTTTTAAA GACCGTAAAG AAACAGAAGC

CTTTACGGTC TTTAAAAAGG CCGTAATATC CAGCTGAACG GTCTGCTGAT

AGGTAGATTG AGCAACTGAC TGAAATGCCT

1320~

ACAAGTTTTA TCCGGCCTTT ATTCACATTC TTGCCCGCCT GATGCAGGCT

CATCCGG

AATTCCGGAT GAGCCTGCAT CAGGCGGGCA AGAATGTGAA TAAAGGCCGG

ATAAAACTTG TGCTTCTGTT T

B. Construction of pTrpCAT72 Q6S3 Starting with pCAT72 Q3Sl, pCAT153 Q6S3 was constructed to change the Asn residues at positions 130, 141 and 148 of CAT to Gln residues, and to change the Cys residues at 91 and 126 to Ser residues.
Pla~mid CAT72 Q3Sl in pUC-9 was clea~ed with EcoRI. Oligos CAT153-1 through 6 (below) were annealed and ligated into pCAT72 to ~ive pCAT153 Q6S3. The modified pCAT153 was then removed from pUC by cleavage with NdéI and HindIII, and the resulting fragment inserted into pTrp233 to give pTrpCAT153 Q6S3.

AATTTCGTAT GGCAATGAAA GACGGTGAGC TGGTGATATG GGATAGTGTT

CACCCTTCTT ACACCGTTTT CCATGAGCAA

~ 10 20 30 40 50 AAA~CGGTGT AAGAAGGGTG AACACTATCC CATATCACCA GCTCACCGTC

TTTCATTGCC ATACGA

~ 10 20 30 40 50 ACTGAAACGT TTTCATCGCT CTGGAGTGAA TACCACG~CG ATTTCCGGCA
~ ~0 70 80 GTTTCTACAC ATATATTCGC AAGATGTGGC

~37~ 132~62 AGCGATGAAA ACGTTTCAGT TTGCTCATG~

-10 ~0 30 40 50 GTCTTACGGT GAACAGCTGG CCTATTTCCC TAAAGGGTTT ATTGAGCAGA

TGTTTTTCG~ CTCAGCCCAG CCCG
CAT153~6 - ~ 10 20 30 4~ 5) AATTCGGGCT GGGCTGAGAC ~A~AAACATC TGCTC~AT~ ACCCTTTA~G
7~ 80 GAAATA~GCC AGCTGTTCAC CGTAAGACGC CACATCTT
~, ~

Next, the human adip~in/D.cDNA hg31-40 (Figure 10) wa~ constructed. The BamHI-~yI fragment containing th~ mature coding region was gel puri~iod and in3erted into pUC-9 which had been cleaved with BamHI and HlndIII.
The StyI end of the cDNA ~a~ ~oined to the HindIII end of pUC using two oligos (#3886 S'-CATGGGTGCCGGGGCCTGA-3~ and #3887 5'-AGCTTCAGGCCCCGGCACC-3'). 8y inserting the BamHI-~yI fragment of adipsin/D into pUC in this way, the cod-ing sequence of adipsin/D wa~ placed in fra~e with the EcoRI site of pVC~9. 1~he EcoRI-HindIII fragment of thi~
construct w~s removed from pUC-9 and in~ertsd into pTrpCAT72 between the EcoRI ~ite and the HindIII sites to yield pTrpCRT72:Adipsin/D.
This con~truct gave 10-15~ lavel~ of fusion protein upon induction in ~3110 c~llY.

~odifications of the above de~cribed mode~ for carrying out the invention that are obvious to thoso of skill in the ar~ of molecular biol~gy, protein chemi~try, cell biology, or rela~ed field~ are in~ended to be within the scope of the following claim~.

~1

Claims (15)

1. A method of stabilizing heterologous protein expression in a prokaryotic host comprising:
(a) constructing a hybrid gene comprising in sequential order, a 3' truncated chloramphenicol acetyltransferase (CAT) gene sequence fused in frame with a heterologous gene sequence encoding a mammalian polypeptide selected from the group consisting of amyloid protein A4-751 insert sequence, glucagon-like peptide 1, adipsin/D, lung surfactant protein SP-B and lung surfactant protein SP-C, wherein said polypeptide is normally not recoverable in bacterial expression systems, and wherein said hybrid gene, upon translation, produces a fusion protein in a recoverable yield;
(b) providing a vector for expression of said hybrid gene;
(c) culturing the prokaryotic host transformed with the expression vector; and (d) recovering the fusion protein.

2. The method of claim 1 wherein said prokaryotic host is a bacterial cell.

3. The method of claim 2 wherein said bacterial cell is E. coli.

4. The method of claim 1 wherein said 3' truncated CAT gene sequence enhances the level of heterologous protein present in the total cellular protein.

5. The method of claim 1 wherein the length of the truncated CAT gene sequence encodes a CAT peptide of about 73 to about 210 amino acids.

6. The method of claims 1 or 5 wherein said hybrid gene further comprises a DNA sequence encoding a selective cleavage site located between the CAT gene sequence and the heterologous gene sequence.

7. The method of claim 6 wherein said selective cleavage site is composed of tryptophan, methionine, asparagine-glycine, or glutamic acid.

8. A method of stabilizing heterologous protein expression in a prokaryotic host comprising:
(a) constructing a hybrid gene comprising in sequential order, a 3' truncated chloramphenicol acetyltransferase (CAT) gene sequence encoding a CAT
peptide of about 73 to about 180 amino acids, fused in-frame with a heterologous gene sequence encoding a mammalian polypeptide selected from the group consisting of amyloid protein A4-751 insert sequence, glucagon-like peptide I, adipsin/D, lung surfactant protein SP-B and lung surfactant protein SP-C, wherein said heterologous protein is normally not recoverable in bacterial expression systems, and wherein said hybrid gene, upon translation, produces a fusion protein in a recoverable yield;
(b) providing a vector for expression of said hybrid gene;
(c) culturing the prokaryotic host transformed with the expression vector; and (d) recovering the fusion protein.

9. The method of claim 8 wherein said hybrid gene further comprises a DNA sequence encoding a selective cleavage site located between the CAT gene sequence and the heterologous gene sequence.

10. A bacterial expression vector capable of enhancing the level of expression of non-stable, bacteri-ally produced heterologous polypeptides comprising:
a hybrid gene having in sequential order, a 3' truncated CAT gene sequence linked to a heterologous gene sequence encoding a mammalian polypeptide selected from the group consisting of amyloid protein A4-751 insert sequence, glucagon-like peptide I, adipsin/D, lung surfactant protein SP-B and lung surfactant protein SP-C, wherein said polypeptide is normally not recoverable in bacterial expression systems, whereby said truncated CAT
gene sequence is capable of rendering the resulting fusion protein resistant to proteolytic degradation.

11. The expression vector of claim 10 wherein the length of the truncated CAT gene sequence encodes a CAT
peptide of about 73 to about 210 amino acids.

12. The bacterial expression vector of claims 10 or 11 wherein said hybrid gene further comprises a DNA
sequence encoding a selective cleavage site located between the CAT gene sequence and the heterologous gene sequence.

13. The vector of claim 12 wherein the hybrid gene having said 3' truncated CAT gene sequence, upon expression, enhances the level of the heterologous protein present in the total cellular protein.

14. In a bacterial expression vector capable of enhancing the level of expression of non-stable, bacteri-ally produced heterologous polypeptides wherein the vector comprises a hybrid gene having in sequential order, a 3' truncated CAT gene sequence linked to a heterologous gene sequence encoding a polypeptide normally not recoverable in bacterial expression systems, said truncated CAT gene sequence being capable of rendering the resulting fusion protein resistant to proteolytic degradation, the improvement comprising altering one or more DNA codons of the truncated CAT gene to eliminate potential chemical cleavage sites within the CAT protein.

15. The improved bacterial expression vector of claim 14 wherein the alterations include substituting the DNA encoding a) methionine at position 67 of CAT with DNA
encoding isoleucine or leucine; (b) cysteine at position 31 of CAT with DNA encoding serine; or (c) tryptophan at position 16 of CAT with DNA encoding tyrosine.