| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 05.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 30.05.00 |
| PATENT TITLE |
High expression Escherichia coli expression vector |
| PATENT ABSTRACT |
High expression vectors for expression of heterologous genes in Escherichia coli. The expression vectors contain, the tac promoter, an intergenic region, a restriction site, and, optionally, groES DNA. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 04.12.98 |
| PATENT REFERENCES CITED |
Castanie et al., Analytical Biochemistry 254(1):150-152 (1997). Lee et al., J. Biol. Chem. 267(5):2849-2852 (1992). Weiss et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp 6019-6023, Oct. 1984. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An expression vector comprising: (a) tac promoter, (b) groESL integenic region of DNA, (c) the start codon of the groEL gene sequence, (d) a restriction site and, (e) a heterologous gene sequence; provided that said expression vector contains no more than the first seven codons of the groEL gene. 2. The expression vector of claim 1 further comprising groES DNA. 3. The expression vector of claim 1 further comprising the first seven codons of groEL and wherein the RsaI site is modified to contain a restriction site different from the RsaI restriction site. 4. The expression vector of claim 1 wherein the restriction site is introduced immediately before the start codon of the groEL gene. 5. The expression vector of claim 1 further comprising an origin of replication and a DNA sequence which encodes a selectable marker. 6. The expression vector of claim 5 wherein the selectable marker is antibiotic resistance. 7. The expression vector of claim 5 wherein the selectable marker is neomycin resistance. 8. The expression vector of claim 1 further comprising sequences coding for the lac repressor. 9. The expression vector of claim 1 having all of the identifying characteristics of plasmids pBMS1999, pBMS2000, or pBMS2001. 10. A prokaryotic host cell containing an expression vector comprising: (a) tac promoter, (b) groESL integenic region of DNA, (c) the start codon of the groEL gene sequence, (d) a restriction site and, (e) a heterologous gene sequence; provided that said expression vector contains no more than the first seven codons of the groEL gene. 11. The prokaryotic host cell of claim 10 wherein said expression vector further comprises groES DNA. 12. The prokaryotic host cell of claim 10 which is E. coli. 13. The E. coli host cell of claim 12 wherein the expression vector further comprises an origin of replication and a DNA sequence which encodes a selectable marker. 14. The E. coli host cell of claim 12 wherein the selectable marker is neomycin phosphotransferase. 15. The E. coli host cell of claim 10 wherein the expression vector has all of the identifying characteristics of plasmids pBMS1999, pBMS2000, or pBMS2001. 16. An E. coli strain having the designation ATCC 98563. 17. A method for expressing a heterologous protein is a prokaryotic host cell comprising culturing a prolkaryotic host cell containing an expression vector comprising: (a) tac promoter, (b) groESL integenic region of DNA, (c) the start codon of the groEL gene sequence, (d) a restriction site and, (e) a heterologous gene sequence; provided that said expression vector contains no more than the first seven codons of the groEL gene. 18. The method of claim 17 wherein said expression vector further comprises groES DNA. 19. The method of claim 17 wherein the prokaryotic host cell is E. coli. 20. The method of claim 17 wherein the expression vector further comprises an origin of replication and a DNA sequence which encodes a selectable marker. 21. The method of claim 20 wherein the selectable marker is antibiotic resistance to neomycin sulfate. 22. The method of claim 17 wherein the heterologous protein is glutaryl cephalosporin amidase or penicillin G amidase. 23. The method of claim 17 wherein the expression vector further comprises sequences coding for the lac repressor. 24. The method of claim 17 carried out at a temperature of about 25.degree. C. to about 37.degree. C. and a pH of about 7.0 to about 7.2. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention concerns high expression vectors for expression of heterologous genes in bacteria such as Escherichia coli. BACKGROUND OF THE INVENTION The ability to express large quantities of recombinant proteins is desirable in many applications and of economical necessity for many industries. This is especially true for products that are of low value, products with small profit margin, technical enzymes (e.g., protease and lipases for detergents) and proteins for in vivo diagnostics (cholesterol oxidase, penicillin-G acylase). There are several expression systems available for the expression of heterologous genes. However, the most valuable, versatile and perhaps the best system for the expression of heterologous proteins is Escherichia coli. There are many publications on the essential elements needed to express heterologous proteins in high levels. One of the most essential elements is the promoter used to express the heterologous genes. The promoter used should be strong. Some of the more frequently used strong promoters for the expression of heterologous genes are the promoters from P.sub.L, tac, trp, trc and the T7 promoter described by Studier et al. The promoters used are generally regulatable. This feature is essential if the target protein to be expressed is toxic to the host. In general, the stronger the promoter, the more RNA will be transcribed from the DNA leading to the accumulation of messenger RNA. Besides strong regulatable promoters, other elements are also involved in the expression of heterologous genes. The efficiency of the translation is involved in maximizing the expression of heterologous genes. The efficiency of translation can be affected by the mRNA 5'-terminus sequences as well as by the 5' end hairpin structure of the mRNA. Generally, a functional ribosome binding site containing a Shine-Dalgarno (SD) sequence properly positioned to an AUG initiation codon is essential for efficient translation. Variation in the distance between the SD sequence and the AUG codon are known to affect mRNA translation. Studies have also shown when the SD sequence or the AUG initiation codon is sequestered in a double-stranded region of the mRNA, translation is less efficient due to the blocking of the accessibility of these sequences to the ribosome. Some other factors that have been reported to affect the efficient expression of heterologous genes are the stability of the messenger RNAs, the susceptibilities of the protein products to proteolysis and the effect of the host genetic background. Although there is a wealth of information about the elements that affect the overall efficiency of a plasmid based expression system, there are other elements that have not been studied which may be involved in the expression of heterologous genes. SUMMARY OF THE INVENTION The present invention is directed to an expression vector comprising: (a) tac promoter, (b) groESL intergenic region of DNA, (c) the start codon of the groEL gene sequence, and (d) a restriction site. In a preferred embodiment the expression vector of the invention further comprises: (e) groES DNA. In another aspect the present invention concerns a prokaryotic host cell containing an expression vector comprising: (a) tac promoter, (b) groESL intergenic region of DNA, (c) the start codon of the groEL gene sequence, (d) a restriction site, and, optionally, (e) groES DNA In another aspect the present invention is also directed to a method for producing a heterologous protein comprising culturing a host cell of the invention under conditions suitable for expression of the protein. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Construction of pBMS1000. FIG. 2. Construction of pBMS1000GroESL. FIG. 3. Construction of pBMS2000H. FIG. 4. Construction of pBMS2000, pBMS2001, pBMS2002. FIG. 5. Construction of pBMS2000.103 and pBMS2000.75. FIG. 6. Construction of pBMS1999GCA and pBMS2000GCA. FIG. 7. Construction of pBMS2000.103GCA, pBMS2000.75GCA, and pBMS2000HGCA. FIG. 8. Construction of pBMS1000PGA, pET9dPGA, and pBMS2000PGA. DETAILED DESCRIPTION OF THE INVENTION The Escherichia coli GroES and GroEL are chaperone proteins which mediate the correct folding of a wide variety of polypeptides and facilitate oligomeric protein assembly by preventing premature interior intramolecular interactions that can lead to aggregation or misfolding structure. The GroES and the GroEL proteins are transcribed from the same mRNA. The expression vectors of the invention are based on the GroESL operon. Expression vectors of utility in the present invention are often in the form of "plasmids", which refer to circular double stranded DNA loops which, in their vector form, are not bound to the chromosome. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto. The vectors of the invention are capable of expressing heterologous genes in large quantities in Escherichia coli. In addition to other features, the vectors of the invention preferably have an origin of replication and a nucleic acid sequence coding for a selectable marker. The particular selectable marker used is not critical provided the marker allows for phenotypic selection in transformed host cells. Preferred selectable markers are antibiotic resistance. Examples of antibiotic resistance markers include ampicillin, tetracycline, chloramphenicol, kanamycin, gentamycin, nalidixic acid, rifampicin, spectinomycin, streptomycin, neomycin phosphotransferase, and the like. In the vectors of the invention, the tac promoter is followed by (i.e., upstream of) the complete gene sequence of the groES gene, which is followed by the intergenic region between the termination codon of the groES gene which is followed by the start codon of the groEL gene (termed herein the "groESL intergenic region"), which is followed by a restriction cloning site. The restriction cloning site can be introduced immediately at the start codon of the groEL gene or at the Rsal site in the coding region of the the groEL gene. Heterologous gene cloned into the former expression vectors will be expressed as the native protein whereas heterologous gene cloned into the latter expression vectors will be expressed as a fusion protein to the first 7 amino acids of the groEL gene. The cloning restriction sites supply the codon, ATG, the codon needed for the initiation of the translation. The vectors of the invention can be categorized into two classes, those containing the groES gene and those without the groES gene. The first class of vectors contain the strong regulatable promoter, tac, followed by the codons coding for the gene GroES. In the vectors of the invention a cloning site is introduced into the Rsal site of the gene coding for GroEL or a cloning site is introduced immediately before the start codon of the GroEL gene. As stated above,in the former case, the gene product will be a fusion protein containing approximately 7 amino acids of the GroEL gene at the amino terminus; in the latter case, the gene product will be the native protein containing the amino acid methionine at the amino terminus. In both types of constructs, all the GroEL sequences after the introduced restriction site preferably are removed. In the second type of vectors, the vectors contain the tac promoter followed by various length of the intergenic sequences between the groES and groEL gene and a restriction site which supply the initiation codon for the cloning of heterologous genes. The groES gene has been eliminated. The deletion of the groES gene sequences can be advantageous in some cases. The co-expression of the groES protein in large quantities may interfere with downstream processing, such as the immobilizaton of the enzymes to solid supports. However, in other instances it may be advantageous to use an expression vector containing the gene sequences for the groES gene (e.g., pBMS2000) because it can stabilize the transcripts and the presence of groES may stabilize the heterlogous protein expressed. The vectors of the invention also optionally contain sequences coding for the lac repressor to regulate the transcription from the lac promoter; this allows expression of the heterologous gene cloned to be controlled by isopropyl-.beta.-D-thiogalactopyranoside (IPTG) or lactose. In a variation of the above mentioned expression vectors, vectors contemplated herein optionally express heterologous genes constitutively. This is accomplished by removing the operator sequences from the promoter region. An example of such a vector is plasmid pBMS2000H. Advantages in using a constitutive expression vector include elimination of the need to add an inducer to induce the system to express the heterologous gene products. This can decrease the cost of goods and simplify fermentation process by eliminating one fermentation parameter needed to be examined such as the optimal IPTG concentration and the optimal time to add the inducer to yield optimal expression of the gene product. It is possible, in some cases, that the cloning of heterologous genes in constitutive expression vectors will yield more heterologous gene products as the gene products are accumulated from the very beginning of the cell growth. The regulatory DNA sequences of the vectors of the invention, such as the promoter and repressor, are operatively linked to the DNA sequence coding for all or part of the heterologous gene sequence desired to be expressed. As used in this context, the term "operatively linked" means that the regulatory DNA sequences are capable of directing the replication and/or the expression of the DNA sequence coding for all or part of the protein desired to be expressed. The vectors of the invention can be used to express a wide variety of heterologous genes. Examples of heterologous genes that can be expressed using the vectors of the invention include D-amino acid oxidase gene from Trignopsis variabilis; genes encoding immunotoxins such as G28.5sFv-PE40 and BR110sFv-PE40; the gene coding for penicillin G amidase, the gene coding for the glutaryl cephalosporin amidase (GCA), and the like. As shown in the Examples section, the vectors of the invention yield higher titers of expressed enzymes (e.g., GCA), relative to other vectors known in the art, such as T-7 RNA polymerase based pET vectors. The expression vectors of the invention may also include other DNA sequences known in the art, for example, stability leader sequences which provide for stability of the expression product, secretory leader sequences which provide for secretion of the expression product, stability elements which provide mitotic stability to the plasmid, and other sequences which provide additional sites for cleavage by restriction endonucleases. The characteristics of the actual expression vector used must be compatible with the host cell which is to be employed. The sequence for the tac promoter is described in, for example, Weiss et al. Proc. Natl. Acad. Sci. U.S.A. 81, p. 6019-6023, 1984. Suitable expression vectors containing the desired coding and control sequences may be constructed using standard recombinant DNA techniques as taught herein or as known in the art, many of which are described in T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982. The present invention additionally concerns host cells containing an expression vector of the invention. Suitable host cells are prokaryotic cells which are preferably biologically pure. Suitable prokaryotic host cells include, for example, Escherichia coli, Bacillus subtilus and Salmonella typhimurum cells. The most preferred host cell of the invention is E. coli DH5.alpha. mcr which is also designated ATCC 98563. E. coli ATCC 98563 was deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 on Oct. 28, 1997, under the provisions of the Budapest Treaty. E. coli ATCC 98563 contains plasmid pBMS2000. Expression vectors may be introduced into host cells by various methods known in the art. For example, transfection of host cells with expression vectors can be carried out by the calcium phosphate precipitation method. However, other methods for introducing expression vectors into host cells, for example, electroporation, biolistic fusion, liposomal fusion, nuclear injection, viral or phage infection or protoplast fusion, can also be employed. Once an expression vector has been introduced into an appropriate host cell, the host cell may be cultured under conditions permitting expression of the desired protein or polypeptide. Host cells containing an expression vector of the invention may be identified by one or more of the following six general approaches: (a) DNA-DNA hybridization; (b) the presence or absence of marker gene functions; (c) assessing the level of transcription as measured by the production of hTOPI mRNA transcripts in the host cells; (d) detection of the gene product immunologically; (e) complementation analysis; and (f) enzyme assay, enzyme assay being the preferred method of identification. In the first approach, the presence of a DNA sequence coding for the desired heterologous protein can be detected by DNA-DNA or RNA-DNA hybridization using probes complementary to the DNA sequence. In the second approach, the recombinant expression vector host system can be identified and selected based upon the presence or absence of certain marker gene functions (e.g., thymidine kinase activity, resistance to antibiotics, uracil prototrophy, etc.). A marker gene can be placed in the same plasmid as the DNA sequence coding for the heterologous gene under the regulation of the same or a different promoter used to regulate heterologous protein production. Expression of the marker gene in response to induction or selection indicates expression of the DNA sequence coding for the heterologous protein. In the third approach, the production of heterologous gene mRNA transcripts can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blotting or nuclease protection assay using a probe complementary to the RNA sequence. Alternatively, the total nucleic acids of the host cell may be extracted and assayed or hybridization to such probes. In the fourth approach, the expression of the heterologous gene can be assessed immunologically, for example, by Western blotting. In the fifth approach, the expression of the heterologous gene can be assessed by complementation analysis. For example, in cells known to be deficient in an enzyme of interest, expression of enzyme activity can be inferred by improved growth of cells under growth-limiting conditions. In the sixth approach, expression of heterologous DNA can be measured by assaying for enzyme activity using known methods. For example, the assay described in Y. Pommier, J. Biol. Chem., 265, pages 9418-9422, 1990 may be employed. The DNA sequences of expression vectors, plasmids or DNA molecules of the present invention may be determined by various methods known in the art. For example, the dideoxy chain termination method as described in Sanger et al., Proc. Natl. Acad. Sci. USA, 74, pages 5463-5467, 1977, or the Maxam-Gilbert method as described in Proc. Natl. Acad. Sci. USA, 74, pages 560-564, 1977 may be employed. It should, for course, be understood that not all expression vectors and DNA regulatory sequences will function equally well to express the DNA sequences of the present invention. Neither will all host cells function equally well with the same expression system. However, one of ordinary skill in the art may make a selection among expression vectors, DNA regulatory sequences, and host cells using the guidance provided herein without undue experimentation and without departing from the scope of the present invention. The present invention is also directed to a method for producing a heterologous protein comprising culturing a host cell of the invention under conditions suitable for expression of the protein. Growth of the host cells may be achieved by one of ordinary skill in the art by the use of an appropriate medium. Appropriate media for growing host cells include those which provide nutrients necessary for the growth of the cells. A typical medium for growth includes necessary carbon sources, nitrogen sources, and trace elements. Inducers may also be added. The term "inducer", as used herein, includes any compound enhancing formation of the desired protein or peptide. Carbon sources may include sugars such as glucose, sucrose, lactose, galactose, raffinose, and the like; nitrogen sources include yeast extract, casamino acids, N-Z amine, bacto-tryptone, and the like. A preferred medium comprises 2.4% yeast extract, 1.2% Bacto-tryptone, 0.4% glycerol, 0.72M dipotassium hydrogen phosphate and 0.17M potassium dihydrogen phosphate. The pH of the medium is preferably adjusted to about 6.8 to 7.5, more preferably about 7.0. The process of the present invention is performed under conditions suitable for expression of the desired peptide. The pH of the medium is preferably maintained between about 7.0 and 7.5, most preferably between about 7.0 and 7.2, during the growth of host cells. A suitable temperature range for the process of the invention is from about 25.degree. C. to about 37.degree. C. Pressure is not known to be critical to practice of the invention and for convenience about atmospheric pressure is typically employed. The process of the invention is preferably carried out under aerobic cond |
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