EXPRESSION VECTORS

Abstract: Provided are expression vectors having ColE1-type replication systems and having mutations in the RNA II gene or translation initiation region of the rop gene, or both, that increase expression of heterologous polypeptides encoded thereby without substantially increasing copy number.

Description: EXPRESSION VECTORS FIELD OF INVENTION This invention relates to improved expression vectors. The expression vectors of this invention are characterized by increased expression of heterologous genes inserted therein as a result of site-specific mutations. BACKGROUND OF THE INVENTION A number of approaches have been taken to maximize heterologous gene expression in hosts transformed with such genes. Although the approaches used may vary for the expression of different genes of interest, in general they involve manipulating expression control sequences regulating expression of the heterologous gene, the DNA sequence of the heterologous gene itself, the expression vector, and the host. The expression control sequences involved include both transcriptional (promoters, operators and terminators) and transla- tional initiation and termination regions. Changes in the cloned heterologous gene include altering the coding sequence to optimize codon usage, to enhance mRNA stability, to disrupt secondary struc¬ ture which might interfere with efficient translation, and to enhance the stability or activity of the protein product (e.g., small unstable proteins are often cloned as tandem repeats to alleviate product stability problems) . Manipulations of the expression vector are usually aimed at building a small, stable plasmid vector, including providing increased dosage of the heterologous gene by increasing plasmid copy number. Alterations in hosts include, for example, mutations that lower the amounts of cytoplasmic proteases produced by the hosts. INFORMATION DISCLOSURE Expression vectors related to ColEl-type plasmids have been used extensively for expression of foreign genes in E. coli because of their small size, genetic stability and relatively high copy number (about 10-15 per cell). One such plasmid, pBR322, is one of the most widely used E. coli vectors. pBR322 has a ColEl-type replication system which was derived from the ColEl plasmid relative pMBl (Bolivar, F. , et al. (1977) Gene, 2, 75-93; Bolivar, F. , et al. (1977) Gene, 2, 95-113). A model for the control of ColEl-type replication systems exists (for reviews, see Davison, J. (1984) Gene, 28, 1-15 and Polisky, B. , in Reznikoff, W. and Gold, L. (eds.) H-zximizing Gene Expression (1986) Butterworth Publishers, Stoneham, MA, 143-170). In summary, an RNA primer transcript (called "primer" or "RNA II") is initiated about 555 bases upstream from the origin of replication of the ColEl- type plasmid by RNA polymerase. As transcription proceeds to the origin, this RNA forms a hybrid with the opposite DNA strand in the vicinity of the origin and extending downstream from it. For successful replication initiation, the hybridized primer must then be cleaved at the origin by ribonuclease H. This processed-RNA II transcript then serves as a primer for replication of the plasmid. The interactions of the primer that lead to replication appear to be controlled in tr-ans by two plasmid encoded molecules. One of these molecules, RNA I, is a 108 to 110 base RNA molecule that is tran¬ scribed from the same DNA as the beginning of RNA II, but in the opposite direction. Thus, RNA I is complementary to the 5' end of RNA II. Through a series of base pairing interactions, RNA I can hybridize to the primer resulting in an RNA II molecule that cannot be processed by RNAse H and thus, can no longer serve as a primer for replication. The interaction between RNA I and the primer represses replication and appears to be responsible for copy number control and the incompatibility behavior observed with ColEl-type plasmids. The other plasmid encoded molecule involved in this process is a 63 amino acid protein. The gene encoding the protein is about 500 base pairs downstream from the origin of replication. This protein, called rop or rom has been shown in vitro to facilitate the interaction of RNA I and the primer, further contributing to the repression of replica¬ tion. Knowledge of ColEl-type replication system control and the use of specific mutations in the control regions have led to the con- struction of a number of high copy number (in some cases regulatable) expression vectors (Boros, I., et al. (1984). Gene, 30, 257-260; Gayle III, R.B. , et al. (1986) Gene, 41, 281-288; EPA 0 013 830; UK patent application 2136814A; PCT application PCT/US82/00246; EPA 0 096 586; PCT/GB85/00110) . Constitutive high copy number mutants (both dominant and recessive) have been isolated and mapped to specific base changes in the RNA I and RNA II structural genes (Muesing, M. , et al. (1981) Cell, 24, 235-242). These mutants affect the structural interactions of RNA I and the primer, thus interfering with replication control. Although point mutations in the rop structural gene, have not been reported, deletion and insertion mutants have been obtained which remove or inactivate the rop gene and thereby produce increased copy number (Twigg, A.J. and Sherratt, D. (1980) Nature, 283, 216- 218; Cesareni, G. , et al. (1982) Proc. Natl. Acad. Sci. U.S.A., 79, 6313-6317; Som, T. and Tomizawa, J. (1983) Proc. Natl. Acad. Sci. U.S.A., 80, 3232-3236; Lupski, J.R. , et al. (1986) Proc. Natl. Acad. Sci. U.S.A., 83, 7381-7385; Nugent, M.E., et al. (1986) J. Gen. Microbiol., 132, 1021-1026). The cDNA coding for mature bovine growth hormone (bGH; bovine somatotropin, bSt, is sometimes also used herein) is expressed poorly in E. coli in typical pBR322-derived plasmids. Expression of bGH in such a system has been enhanced by various methods. Seeburg, et al. (1983) DNA, 2, 37-45, and George, et al. (1985) DNA, 4, 273-281, have increased expression of bGH in such a system by replacing the natural coding sequence corresponding to the first 24 amino acids of bGH. Some of these changes were made to reflect the E. coli codon usage bias while others were made to disrupt potential secondary structure that could form in the RNA. In another approach, a "two-cistron" construct of the gene was made (Schoner, B.E., et al. (1984) Proc. Nat. Acad. Sci., U.S.A., 81, 5403-5407; Schoner, B.E., et al. (1986) Proc. Natl. Acad. Sci., U.S.A., 83, 8506-8510). In this case, the first cistron, 31 base pairs long, consisted of an ATG start codon followed by a short A-T rich region containing two cloning sites, a Shine-Dalgarno sequence, another small A-T rich spacer, a TAA stop codon, and another ATG start codon connected to the bGH cDNA. This construct produced high bGH expression from a plasmid with a ther oindueible runaway replicon. Schoner, et al. attributed the increased expression to increased translation due to overcoming secondary structure problems near the 5' -end of the mRNA. (See also, EPA 111,814) The present invention addresses the problem of poor expression of bGH and other heterologous cDNAs from expression vectors having a ColEl-type replication system in E. coli and other related hosts. To alleviate these problems, we created random point mutations in a pBR322-derived plasmid which contained a good E. coli promoter and translation initiation region upstream of a bGH cDNA that contained one amino acid change from that encoded by the native bGH cDNA and operatively linked thereto. We chemically mutated purified covalen- tly closed circular plasmid DNA from expression vectors containing bGH cDNA constructs and screened E. coli transformed with this DNA for increased bGH expression. These vectors are also useful for expressing other somatotropins (e.g., porcine and ovine) as well as other heterologous polypeptides (e.g., interleukin-1) . The instant invention provides novel expression vectors for expression of heterologous polypeptides in E. coli and other related hosts. High expression is achieved without substantially increasing (e.g., less than a three-fold increase) copy number at physiological temperature. The increased expression is due to novel mutations in pBR322-specific portions of the vectors in regions known to affect control of replication. The point mutation in the specific expres- sion vector that we designated pMbSt30 is in the promoter region of RNA I and also, therefore, in the structural gene for the counter transcript, RNA II, the primer of replication. The only prior known mutations reported in the RNA I promoter (Wong, E.M. , et al. (1982) Proc. Natl. Acad. Sci. U.S.A., 79, 3570-3574) led to a temperature sensitive copy number phenotype which was attributed to a change in the RNA II structure. The point mutation In our pMbSt31 expression vector is in the Shine-Dalgarno region of the rop gene. This mutation is novel and unique; no point mutations in the rop gene or its regulatory sequences have been reported until now. We have also made a third expression vector, pMbStl, comprising both of these mutations. SUMMARY OF THE INVENTION The present invention relates to an expression vector having a ColEl-type replication system, comprising a mutation in the RNA II gene, or the translation Initiation region of the rop gene, or both, and a restriction site wherein a DNA sequence encoding a heterologous polypeptide may be inserted into the vector and operatively linked therein to an expression control sequence, said mutations not substantially increasing the copy number of the expression vector. More specifically, the vector is a pBR322-derived vector. More specifically, the heterologous polypeptide is bGH. Still more specifically, the expression vector is selected from pMbSt30, pMbSt31, pMbStl, and derivatives thereof. Also provided are hosts transformed with the vectors and methods for expressing the heterologous polypeptides comprising culturing the transformed hosts. DETAILED DESCRIPTION OF THE INVENTION As used herein, "expression control sequence" refers to a sequence of nucleotides that controls and regulates expression of genes when operatively linked to those genes. It includes the promoter, translation initiation region (e.g., ribosome binding sites) and other sequences useful in the expression of genes that are operatively linked to those expression control sequences. The term "operatively linked" includes having an appropriate start signal in front of the gene encoding the desired product and maintaining the correct reading frame to permit expression of the inserted gene under the control of the expression control sequence and synthesis of the desired product encoded for by that gene. The "translation initia¬ tion region" is that portion of the DNA molecule located upstream of the gene encoding the desired product that affects efficiency of translation initiation and includes, for example, the Shine-Dalgarno sequence. Somatotropins (growth hormones) are well known to those skilled in the art and include porcine, ovine and bovine somatotropins. Several analogs of the naturally occurring somatotropins are also known. PCT patent application No. PCT/US88/00328 sets forth many of. these analogs and is incorporated herein by reference. (See also, Seeburg, et al. , supra and George, et al., supra.) Other hetero¬ logous polypeptides include those polypeptides not naturally expressed by the host organism. Any of a large number of available and well known host cells may be used in the host-expression vector combinations of this invention. The selection of a particular host is dependent upon a number of factors recognized in the art. These include, for example, com¬ patibility with the chosen expression vector, toxicity of the proteins encoded for by the hybrid plasmid, ease of recovery of the desired protein, expression characteristics, biosafety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence in the expression vectors and methods of this invention. Wi hin these general guidelines, useful hosts may include bacteria, such as strains of E. coli , Salmonella and other hosts in which the vectors can replicate. Wild-type E. coli strain K12 is the preferred bacterial host strain and was purchased from the American Type Culture Collection. Plasmids were constructed as follows: pLG23, a plasmid contain¬ ing cloned cDNA for bGH, was obtained from Fritz Rottman (Case Western Reserve University) . This plasmid was deposited on 12 May 1981 at the Northern Regional Research Laboratory, Peoria, Illinois, and assigned accession number NRRL B12436. Construction of the plasmid is fully described in European patent application 67026, published 15 December 1982. pSK4, an expression vector containing the trp promoter region, was previously described (Kaytes, P.S., et al. (1986) J. of Biotech., 4, 205-218).). Plasmid pSK106 (see Chart A) was constructed by cloning a synthetic linker (containing an ATG start codon with an overlapping TAA stop codon, a nucleotide bias maximized "spacer" region, and nucleotides to regenerate a Clal site) into the Clal site of pSK4 along with the Klenow treated Haell/Bglll fragment containing the bGH cDNA from pLG23. The synthetic linker creates a phe (TTC) instead of an ala (GCC) codon for the first amino acid in the bGH cDNA. Other pBR322-derived plasmids also can be used, as can others having similar ColEl-type replication systems (e.g., pl5A, Chang, A.C.Y. and Cohen, S.N. (1978) J. of Bacteriology, 134, 1141-1156; Selzer, G. , et al., (1983) Cell, 32, 119-129). Transformants were selected on amp plates (Antibiotic Medium #2 (Difco) containing 50 μg/ml ampicillin) . LB (Luria-Bertanl) medium and M9 medium have been described (Maniatis, T. , et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labora¬ tory, Cold Spring Harbor, New York; Davis, R.W. , et al. (1980) Advanced Bacterial Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Trp starvation plates contained M9 medium supplemented with 0.2% glucose, 1 μg/ml thia ine and 50 μg/ml ampicillin. Trp Induction medium was liquid M9 containing 0.2% glucose, 1 /--g/ml thiamine, 0.05% casa ino acids (acid hydrolyzed; Difco), and 50 /ig/ml ampicillin. Plasmid DNA minipreps were prepared by the method of Birnboim, H.C. and Doly, J. (1979) Nucleic Acid Res. , 7, 1513-1523. Transfor¬ mations were carried out according to Morrison, D.A. (1977) J. Bacteriol., 132, 349-351, or the manuf cturer's instructions. Puri¬ fication of plasmid DNA on CsCl gradients, restriction endonuclease digestions, agarose gel electrophoresis, purification and elution of fragments, and other recombinant DNA techniques were done according to Maniatis et al., supra. Restriction endonucleases, Hindlll lambda DNA markers, T4 DNA ligase and Klenow fragment were obtained from New England Biolabs and used according to their recommendations. Calf intestinal alkaline phosphatase (cat. no. 713023) was purchased from Boehringer-Mannheim. Avian myeloblastosis virus (AMV) reverse transcriptase was obtained from Seikagaku American, Inc. Protein molecular weight markers, ultra-pure (enzyme grade) urea, nick translation reagents, and deoxy- and dideoxynucleotides were from Bethesda Research Labs. Cesium chloride, acrylamide, N,N' -diallyltartardiamide, and antibody screening reagents (Tween 20, protein A-horseradish peroxidase conjugate, and HRP color reagent) were bought from BioRad. Sodium nitrite was obtained from Mallinkrodt. BSA (bovine serum albumin), gelatin, agarose, proteinase K, ribonuclease A, and lysozyme were purchased from Sigma. Deoxyribonuclease I (from bovine pancreas) was from Pharmacia. Alpha and gamma ^^p.^eoxycytidine triphosphate and 5' - 3 H-thymidine were from Amersham. "S-L-methionine was obtained from NEN Research Products (DuPont) . Purified bGH and anti-bGH polyclonal rabbit antibody were made at The Upjohn Company by well- known methods. Immunological screening for bGH expression was done according to a modified combination of a BRL Focus (Vol. 6, No. 1, p.1-5, 1984) and the Bio-Rad Immun-blot Goat Anti-Rabbit (GAR) or Goat Anti-Mouse (GAM) Horseradish Peroxidase (HRP) Conjugate Instruction Manual. A similar procedure was published by Wang, S. Z. and Esen, A. (1985) Gene, 37, 267-269. Competent cells were transformed with mutagenized plasmid DNA (see Example 1) and plated on amp plates at a concentration to give several hundred to several thousand trans- for ant colonies per 100mm plate. After incubation overnight at 37° C, the ampicillin-resistant transformant colonies were lifted on nitrocellulose filters (Millipore, Type HA) and transferred to trp starvation plates for from six hours to overnight at 37" C to induce expression from the trp promoter. The filters were removed from the plates and suspended carefully from a pipet using small binder clamps above about 1.0 mm of chloroform in an enclosed container in a fume hood for one hour to overnight to kill and lyse the cells. The filters then were transferred to several hundred ml of freshly prepared lysis washing and blocking solution containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 3% BSA, 1 μg/ml deoxyribo- nuclease I, and 40 μg/ml lysozyme and incubated with shaking at room temperature for one hour to overnight. The blocking solution was discarded and the filters were washed two times for five minutes with shaking at room temperature in several hundred ml of TTBS (Tween-Tris buffered saline) composed of 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20. The washed filters were incubated four hours to overnight with shaking at room temperature with several hundred ml anti-bGH antibody (a 1/500 dilution in TTBS containing 1% gelatin of anti-bGH, pre-absorbed with E. coli strain K37 extract) . The antibody solution was removed and the filters were washed two times for five minutes with TTBS as before. The filters were then In¬ cubated with several hundred ml of a 1/10,000 dilution in TTBS containing 1% gelatin of Bio-Rad protein A-horseradish peroxidase conjugate for one hour with shaking at room temperature. The filters next were washed two times in TTBS as before and then washed one time in TBS (20 mM Tris-HCl, pH 7.5, 500 mM NaCl). This final wash solution was thoroughly drained from the ilters. Color development solution was prepared freshly on ice by dissolving 180 mg of Bio-Rad HRP color reagent in 60 ml ice cold absolute methanol. 180 μl hydrogen peroxide was added into 300 ml room temperature TBS and mixed. The first solution was added rapidly into the second, mixed, and the combined solution was added to the filters and incubated at room temperature until color was fully developed (30-45 min.). The intensity of the purple color was an indication of the level of bGH expression for a particular clone. Polyacrylamide gel electrophoresis and western blotting were done as follows: Overnight cultures were grown with shaking at 37° C in LB (containing 50 μg/ml ampicillin for plasmid containing strains) and diluted 1/200 in liquid trp induction medium (20 ml) to induce expression of bGH from the trp promoter. The cultures were grown to mid to late log phase (A560 - 0.6-1.0) and harvested when inclusion bodies were clearly visible in high bGH-producing control strains (inclusion body formation indicates high levels of bGH expression and accumulation in the cells) . The cells were harvested by centrifuga- tion and the resulting pellets were suspended to an A560 °f 25.0 per ml in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM Na 2 EDTA) and diluted in TE buffer and 5X protein gel sample buffer (Laemmli, U.K. (1970) Nature, 227, 680-685) to give an A550 of 0.2 per 20 μl . Samples were boiled for three minutes and loaded on SDS-polyacrylamide gels. Pre¬ paration of polyacrylamide gels (5% stacking layer, 17% separation layer) was essentially as previously described (Morse, L.S., et al. (1978) J. Virol., 26, 389-410)-. Gels were run at 30 mA constant current until the BPB dye marker was about 2 cm from the bottom. Duplicate gels were performed for all samples. One of these was subjected to the standard method of Coomassie Brilliant Blue R-250 staining and bGH expression was monitored by scanning densitometry and quantitated as percent of total protein. The duplicate gel was subjected to western blotting (Towbin, H. , et al. (1979) Proc. Natl. Acad. Sci. U.S.A., 76, 4350-4354) and anti-bGH antibody immunological screening as described above. DNA sequencing was performed by the chemical degradation method of Maxam and Gilbert (Maxam, A.M. and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A., 74, 560-564; Maxam, A.M. and Gilbert, W. (1980) Methods Enzymol. , 65, 499-560) and by the dideoxy chain termination method of Sanger et al. (Sanger, F. , et al. (1977) Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467; Sanger, F. and Coulson, A.R. (1978) FEBS Letters, 87, 107-110) as modified by Zagursky, R.L. , et al. (1985) Gene Anal. Tech., 2, 89-94, using double stranded plasmid DNA and AMV reverse transcriptase. Maxam and Gilbert sequencing reaction products were analyzed on denaturing urea-polyacrylamide gel systems described by Slightom, J.L., et al. (1987) J. Biol. Chem. , 262, .7472- 7483. Plasmid copy number was determined by two methods. One method involved scanning densitometry of DNA preparations run on agarose gels in a manner similar to previously reported procedures (Russell, D.R., et al. (1984) Gene, 32, 337-348; Nishi, T. and Itoh, S. (1986) Gene, 44, 29-36; Lupski, J.R., et al. (1986) Proc. Natl. Acad. Sci. U.S.A., 83, 7381-7385; Lin-Chao, S. and Bremer, H. (1986) Mol. Gen. Genet., 203, 143-149; Steuber, D. and Bujard, H. (1982) EMBO Journal, 1, 1399-1404). Cultures were grown as described above in liquid trp induction medium and, immediately prior to harvesting, spiked with an equal volume of E. coli K12 containing pBR322. This served as an internal control for normalization of DNA recovery. Plasmid DNA inlpreps were prepared as described above. Aliquots were digested with RNAse (DNAse free) and EcoRI (to linearize) and sets of dilu- tions were subjected to electrophoresis in 1% agarose gels. The EtBr-stained, ultraviolet-illuminated gels were photographed with Polaroid Type 55 Positive/Negative film and the negatives were scanned with a Shimadzu dual-wavelength chromatogram scanner (Model CS-930). The plasmid peak values in each lane were normalized to the internal pBR322 control plasmid and the copy number ratio was determined by dividing relative molar ratio of the mutant plasmid by that of the unmutagenized plasmid. The other method of plasmid copy number determination was quantitation of DNA from lysates by dye-buoyant density gradient centrifugation (Stougaard, P. and Molin, S. (1981) Anal. Biochem. , 118, 191-193; Womble, D.D., Taylor, D.P. and Rownd, R.H. (1977) J. Bacterial., 130, 148-153; Caro, L., et al. (1984) Meth. Microbiol., 17, 97-122). 10.0 ml cultures of trp induction medium containing 0.02% acid hydrolyzed casamino acids and 50 μg/ml uridine (to facilitate labelling of the DNA; Caro, et al. , supra), 100 μg/ml ampicillin, and 20 Ci/ml 'H-thymidlne were grown in 25 ml DeLong flasks as above. Parallel cultures without label were also grown for monitoring cell growth and inclusion body formation. Cells were harvested at late log-phase by centrifugation and the cell pellets were immediately frozen on dry ice and stored overnight at -80* C. The frozen cell pellets were thawed slowly on ice and each suspended in 1.0 ml 10 mM Tris-HCl, pH 8.0, 50 mM Na 2 EDTA and lysates were prepared as follows. 100 μl of 5 mg/ml lysozyme in OΗ2O was added to each and set at 37° C for 10 minutes. 12.5 μl 20% SDS was added, mixed gently, 100 μl of 20 mg/ml proteinase K was added and incuba¬ tion was carried out for two hours at 37" C.and then 30 minutes at 65" C. The tubes were transferred to room temperature and the chromosomal DNA was sheared by drawing each solution up and down 20 times in 1.0 ml plastic pipets. 0.5 ml of each lysate (2,000,000 to 3,000,000 cpm incorporated into DNA as determined by TCA precipita¬ tion and scintillation counting) was used to prepare 5.5 ml EtBr-CsCl gradients in Beckman quick-seal ultracentrifuge tubes. The samples were spun for 20 hours at 48,000 rpm in a VTi65 rotor. Gradient fractions were collected from the bottom of the centrifuge tubes with a Hoeffer gradient tube fractionator (FS 101) . Successive fractions were collected onto 2 cm X 2 cm Whatman 3MM filter squares until the entire gradient was fractionated (about 120 to 150 samples per gradient) . The filters were washed 3 times with 10% TCA, one time with 100% EtOH, dried under a heat lamp and each counted for tritium in 6 ml Aquasol (DuPont/NEN) . The counts were plotted and the total counts in the plasmid DNA and chromosomal DNA peaks were determined (corrected for background; about 100% of loaded counts were recovered in each gradient) . Based on these counts the relative copy number per chromosome was determined (plasmid DNA peak cpm/chromosomal DNA peak cpm) . The copy number per chromosome was then calculated by dividing these values by the ratio of the molecular weight of one plasmid to the molecular weight of one chromosome (in this case, 3.366 x 10 6 /2.5 x 10 9 - 0.00135). The relative molar ratio was then calculated as above. For in vitro transcription-translation experiments a DNA expression system (procaryotic, in vitro; NEK 038) was purchased from DuPont and used according to the manufacturer's instructions. Each reaction (20 μl total volume) contained 40 μCi 35 S-methionine and 0.5 μg of plasmid DNA which had been purified on two CsCl-EtBr dye- buoyant density gradients. The reactions were terminated by freezing the samples on dry ice. 5.0 μl of 5X protein gel sample buffer (Laemmli, supra) was added to each and the samples were boiled for three minutes. 2.0 μl samples from each fraction were subjected to SDS-polyacrylamide gel electrophoresis and the reaction products were visualized by autoradiography of the dried gel. All of the references cited above under the Detailed Description of the Invention are hereby incorporated by reference. EXAMPLE 1 In vitro mutagenesis of CsCl-purified, covalently closed circular plasmid pSK106 DNA with nitrous acid was carried out by modification of previously published procedures (Fried, M. (1965) Virology, 25, 669-671; Bautz-Freese, E. and Freese, E. (1961) Virology, 13, 19-30; Williams, J.F., et al. (1971) J. Gen. Virol., 11, 95-101; Botstein, D. and Shortle, D. (1985) Science, 229, 1193- 1201). A fresh 1.0 M stock solution of nitrous acid (sodium nitrite) was prepared in ice cold double distilled water and set on ice. Reaction tubes of 1.0 ml containing a final concentration of 0.5 M sodium acetate buffer, pH 4.6, 5 μg to 100 μg CsCl purified plasmid DNA (in up to 130 μl 10 mM Tris-HCl, pH 8.0) and 0.2 ml of 1.0 M fresh nitrous acid stock solution (final concentration 0.2 M) and water were prepared on ice. The mutagenesis reactions were started by shifting the tubes to a 37" C water bath. After 30 minutes the mutagenesis reactions were stopped by adding 2.5 volumes (2.5 ml) 1.0 M Tris-HCl, pH 8.0, to neutralize the nitrous acid. The material then was dialyzed overnight at 4 β C versus two changes of 2.0 liters of TEN buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM Na 2 EDTA) to remove the nitrous acid. The DNA from each reaction was recovered by ethanol precipitation, dried, and dissolved in a final volume of 40 μl 10 mM Tris-HCl, pH 8.0. Usually 10 μl of the resulting muta¬ genized DNA was used to transform 200 μl of competent E. coli cells with selection at 37° C for ampicillin resistance. We isolated the plasmid specific mutations shown in Chart A. Mutations in plasmids from colonies showing higher bGH expression using antibody to bGH as described above were mapped by subcloning restriction fragments back into the unmutagenized plasmid. One high bGH expression mutant plasmid, designated pMbStl, had two mutations by this analysis, both of which mapped outside of the bGH gene region. Two new plasmids, designated pMbSt30 and pMbSt31, each having one of the mutations from pMbStl, were derived by subcloning. The exact location of each of these two mutations was determined by a combination of dideoxy- and Maxam and Gilbert sequencing. The base changes and locations are shown in Chart B. It was found that the mutations in plasmids pMbSt30 and pMbSt31 each increased bGH expres¬ sion as compared to the pSK106 bGH construct. When these two mutations were contained together in the same bGH expression plasmid (i.e., pMbStl) , bGH was expressed at even higher levels. We have observed bGH expression levels in E. coli K12 strains harboring pMbStl of up to 20% of total cellular protein after induction as compared to about 1% for pSK106. The two methods of plasmid copy ntimber determination revealed that there was less than a three-fold copy number increase of plasmid pMbStl in cells induced for expression at physiological temperature (relative copy number, pMbStl/pSK106, was 2.10 by mlniprep gel scanning and 1.15 by counting CsCl gradients). Increased bGH expression from the plasmid mutants was also observed in in vitro transcription-translation assays. These assays further suggest that the increased bGH expression from the plasmid mutants is independent of plasmid copy number. EXAMPLE 2 In this example we set forth the site-specific production of the base changes that were produced by random mutation and selection in plasmids of Example 1 above. Plasmid pMbSt30 is constructed according to the published procedures as follows. A synthetic oligonucleotide (oligo 1, 5' GGCTACACTAAAAGGACAGTATTT 3') is hybridized to pSK106 according to the procedure of Schold, et al. (1984) DNA, 3, 469-477. Following the hybridization step, the oligonucleotide is extended in a reaction mix containing reverse transcriptase, gene 32 protein, dNTP's and appropriate buffers as described. The resulting mixture is used to transform competent E. coli cells (JM101 from Stratagene) and plated onto LB plates containing ampicillin. The resulting transformed colonies are lifted onto nitrocellulose and prepared for hybridiza¬ tion as described (Grunstein, M. , and Hogness, D.S. (1975) Proc. Natl. Acad. Sci., 72, 3961-3965). Oligo 1 is 5' -end-labeled with --P-AΥ ~ ? as described (Maniatis, et al. , supra). The nitrocellulose filters are then hybridized with the oligonucleotide probe as described by Goeddel, D.V. , et al. (1981) Nature, 290, 20-26. Following hybridization, the probe containing solution is removed and the filters are washed in 0.1% SDS, 5 X SSC at a temperature where the oligonucleotide washes off preferentially from the wild-type sequence in pSK106 (Wallace, R.B. , et al. (1981) Nucl. Acids Res., 9, 879-894). Plasmid DNA's from the strongest hybridizing colonies are prepared and sequenced to confirm the altered sequence (5' CACTAAAAG- GACA 3') at nucleotide numbers 3708 to 3720. Plasmid pMbSt31 can be constructed using the same techniques as described above using oligo 2 (5' CCCCTACACGGAAGCATCAAG 3') for the mutagenesis and hybridization steps. Plasmid pMbStl can be constructed by purifying the 2064 bp EcoRI-Ndel fragment containing the bla gene from pMbSt30 and ligating it to the purified 3035 bp EcoRI-Ndel fragment containing the bSt gene and rop gene from pMbSt31. The resulting recombinant plasmids can be sequenced to confirm the presence of the mutations. EXAMPLE 3 In this example, we set forth the expression of a bGH analogue in pMbStl (Example 1) . The plasmid pTrp-BStm4 is a bGH expression vector previously described (U.S. patent application No. 016,294). It contains an analog of the bGH cDNA gene in which the codon for the forth amino acid (alanine) was changed from GCC to GCT. The small EcoRI-Mstll fragment of pTrp-BStm4 which contains expression control elements and the 5'-coding region of the gene was used to replace the analogous fragment of pMbStl (the plasmid mutant with both mutations) . E. coli cells transformed with the resulting plasmid showed a 5- to 10-fold Increase (from less than 0.1% to greater than 0.5%) in bGH expression from the analog gene. Increased expression was also observed with several other bGH gene analogs cloned into pMbStl. EXAMPLE 4 The plasmid mutants described here are also useful for the expression of other genes and their analogs. A DNA fragment contain¬ ing the cDNA for human IL-1 was isolated from RNA of the human hepatoma cell line SK-hep-1 and an HgiAI-Pstl fragment containing the mature IL-lj8 coding region was cloned in frame into the Asp718 site of the expression plasmid pTrp-conSD (U.S. patent application No.016,294) as described by Carter, D.B. , et al. (1988) Proteins:Structure, Function and Genetics, in press. This construct results in an analog of the mature IL-lJ cDNA gene which codes for three additional amino acids at the amino terminus of the mature IL- lβ protein (Carter, D.B., et al., supra). This gene analog is refered to as TL-lβ+. Expression of the IL-13+ protein in J _ coli cells containing the resulting plasmid, pTrp-conSD-ILl, was much less than 1% of total cellular protein. The small EcoRI-Sall fragment contain- ing the IL-1 + gene was isolated from pTrp-conSD-ILl and used to replace the analagous fragment (which contained a modified bGH gene) of pMbStl. E. coli cells transformed with the resulting plasmid expressed IL-1 + protein at a 5 to 10 times higher level than cells containing pTr -conSD-IL1. Chart A. Plasmid pSK106 and Location of Novel Mutations EcoRI pMbSt31 pMbSt30 EcoRI *J . I . I L * I I I I 1 > I I I . I I I > I I I I I I I I I ( < ( I I ( 1 ( ( P t _ bGH rop ori RNA2 -H RNAI The asterisks (*) indicate that the single line represents a circular double-stranded DNA molecule. Only the relative positions of the genes, restriction sites, control elements, and mutations are shown. Chart B. Location of Specific Base Changes in pSK106 to Produce Plasmids pMbSt30 and pMbSt31 rop pSK106 CCCCTTACACGGAGGCATCA» » ' ' 2636 J. 2655 ρMbSt31 A RNA 1 pSK106 ACGGCTACACTAGAAGGACA 3701 4. 3720 pMbSt30 A In each case, the vertical arrow indicates the location of the single base pair changes which produced the point mutations in pMbSt31 and pMbSt30. The nucleotide numbers are according to J. G. Sutcliffe, Cold Spring Harbor Symp. Quant. Biol. , 43, pp. 77-90 (1979).

Claims: CLAIMS 1. An expression vector having a ColEl-type replication system, comprising a mutation in the RNA II gene, or the translation initia¬ tion region of the rop gene, or both, and a restriction site wherein a DNA sequence encoding a heterologous polypeptide may be inserted into the vector and operatively linked therein to an expression control sequence, said mutations not substantially increasing the copy number of the expression vector. 2. A vector according to claim 1, wherein the heterologous polypep¬ tide is a somatotropin. 3. A vector of claim 2, wherein the somatotropin is selected from the group consisting of porcine, ovine and bovine somatotropins. 4. A vector of claim 3, wherein the somatotropin is bovine. 5. A vector according to claim 1, wherein the increase in copy number is not more than threefold. 6. A vector according to claim 1, wherein the mutation In the RNA II gene Is also a mutation in the RNA I promoter. 7. A vector according to claim 1, wherein the mutation in the translation initiation region is in the Shine-Dalgarno sequence. 8. A vector according to claim 1, wherein the vector is a pMBl- derived vector. 9. A vector according to claim 8, wherein the vector is a pBR322- derived vector. 10. A vector according to claim 9, wherein the vector is a pSK106- derived vector. 11. The vector of claim 10, wherein the heterologous polypeptide is selected from the group consisting of porcine, ovine and bovine somatotropins. 12. Vector of claim 11, wherein the somatotropin is bovine. 13. The vector of claim 10, selected from pMbSt30, pMBSt31, pMbStl and derivatives thereof. 14. A method for expressing a heterologous polypeptide comprising culturing a host transformed with an expression vector according to claim 1. 15. A host transformed with an expression vector according to claim 1. 16. A host according to claim 15, selected from the group consisting of E. coli and Salmonella. 17. A host according to claim 16, E. coli.

Inventors: OLSON ERIC R, WATSON EDWARD B III, OLSON, ERIC, R., WATSON, EDWARD, B., III
Applicants: UPJOHN CO, THE UPJOHN COMPANY
Application year: 1988
Main classification: C12N15
IPC codes: C07K14, C12N1, C12N15, C12P21, C12R1