EP0093121A4 - Expression der eiweisssynthese in e. coli. - Google Patents

Expression der eiweisssynthese in e. coli.

Info

Publication number
EP0093121A4
EP0093121A4 EP19820902784 EP82902784A EP0093121A4 EP 0093121 A4 EP0093121 A4 EP 0093121A4 EP 19820902784 EP19820902784 EP 19820902784 EP 82902784 A EP82902784 A EP 82902784A EP 0093121 A4 EP0093121 A4 EP 0093121A4
Authority
EP
European Patent Office
Prior art keywords
plasmid
gene
cells
dominant
allele
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19820902784
Other languages
English (en)
French (fr)
Other versions
EP0093121A1 (de
Inventor
Alvin Markovitz
Barbara A Zehnbauer
Joyce M Schoemaker
Marc F Charette
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University Patents Inc
Original Assignee
University Patents Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Patents Inc filed Critical University Patents Inc
Publication of EP0093121A1 publication Critical patent/EP0093121A1/de
Publication of EP0093121A4 publication Critical patent/EP0093121A4/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present invention relates generally to genetic engineering and more specifically to use of recombinant DNA techniques to secure expression of protein synthesis in selected unicellular organisms.
  • microorganisms have provided a source of commercially significant products such as antibiotics, enzymes and other biologically active proteins, alcohols and the like.
  • Bacteria and yeasts have been the subject of intensive research effort which has had as its goal the enhancement of the cellular and extracellular yield o •*f such commercial products by naturally-occurring and artificially mutated (e.g. , radiation treated) strains of organ- isms. While manipulation of fermentation and product harvesting process parameters can frequently bring about dramatic increases in product yield for selected strains, it is most frequently the case that no degree of modification of culture and isolation conditions will result in enhancing an organism's "expression" of a particular product as evidenced by increased product yields.
  • U.S. Letters Patent No. 4,237,224 to Cohen, et al. relates to transformation of procaryotic unicellular host organisms with "hybrid" viral or circular plasmid DNA which includes exogenous DNA sequences.
  • the procedures of the Cohen, et ⁇ al. patent first involve manufacture of a transformation vector by enzymatically cleaving viral or circular plasmid DNA to form linear DNA strands. Selected foreign DNA strands are also prepared in linear form through use of similar enzymes.
  • the linear viral or plasmid DNA is incubated with foreign DNA in the presence of ligating enzymes capable of effecting a restoration process, and "hybrids" are formed which include the selected foreign DNA segment "spliced" into the viral or circular DNA plasmid. Transformation of -compatible host unicellular organisms with the hybrid vector and propogation of transformant cells results in' the formation of multiple copies of the foreign DNA in the host cell population. In some instances, the desired result is simply the amplification of the foreign DNA and the "product" harvested is DNA. More frequently, the goal of transformation is the expression by the host cells of the foreign DNA in the form of large scale synthesis of isolatable quantities of, e.g. , commercially significant protein and polypeptide fragments coded for by the foreign DNA.
  • marker gene sequences Another manipulative "tool” largely re ⁇ sponsible for successes in transformation of pro- caryotic cells is the use of selectable "marker” gene sequences. Briefly put, hybrid vectors are employed which contain, in addition to the desired foreign DNA, one or more DNA sequences which code for expression of a phenotypic trait capable of distinguishing transformed "from non-transformed host cells. Typical marker gene sequences are those which allow a transformed procaryotic cell to survive and propogate in a culture medium containing metals, antibiotics, and like components which would kill or severly inhibit propagation of non-transformed host cells.
  • endogenous and exogenous gene directed expression of protein synthesis by Escherichia coli cells is en- hanced by transformation of the cells to incorporate one or more copies of a dominant mucoid mutant allele of capR (Ion) gene, preferably the capR9 allele.
  • the mutant capR9 allele is publicly available as a com ⁇ ponent of the DNA plasmid, pBZ20lM9 (A.T.C.C. Plasmid No. 40041) which plasmid provides a presently pre ⁇ ferred vehicle for effecting ⁇ __ coli cell transforma ⁇ tion according to the invention.
  • the ex ⁇ pression of protein synthesis by endogenous genes of naturally occurring and mutant E ⁇ _ coli strains is enhanced by the simple transformation of cells with an E. coli plasmid such as A.T.C.C. Plasmid No. 40041 which includes the capR9 allele.
  • E. coli plasmid such as A.T.C.C. Plasmid No. 40041 which includes the capR9 allele.
  • the same procedure is available to enhance expression of protein synthe- sis in E_ ⁇ coli strains which have had exogenous, protein synthesis directing, genes stably incorporated into chromosomes.
  • E ⁇ _ coli strains which have been transformed with a first vector including a selected exogenous DNA sequence coding for a protein of interest are addi ⁇ tionally transformed with a plasmid providing the capR9 allele such as A.T.C.C. Plasmid No. 40041.
  • the capR9 allele may be incorporated on the same hybrid vector as the selected exogenous DNA sequence and both genes (possibly under the control of the same promotor) may be simultaneously introduced into the ⁇ ]_-_ coli host, with verification of the transformation secured through monitoring for a single marker gene.
  • the term “protein” shall mean and include not only whole functional proteins, but also polypeptides. "Enhancement of expression” of protein synthesis shall designate increasing the net quantity of a selected protein isolatable from a system, whether such increase is attributable to an increase in the amount of the protein synthesized by cells in the system or to an increase in synthesized intact protein remaining available for harvesting from the system.
  • the term “genetically transforming” shall mean effecting the stable incorpo ⁇ ration of 'a selected DNA sequence into an E ⁇ coli cell, whether by way of insertion into chromosomal DNA or by way of incorporation of a vector including the selected sequence.
  • the present invention has its origins, in part, in work commenced over fifteen years ago by co-inventor Markovitz and his co-workers. See, e.g. , Markovitz, et al., P.N.A.S. 5_1, 239 (1964) and 54_ 1084 (1965) . Briefly stated, this work dealt with mutants of an E ⁇ coli K-12 regulator gene, "capR” (also referred to in the art as the "Ion” gene) which controls the synthesis of capsular polysaccharide and the enzymes involved in capsular polysaccharide synthesis.
  • capR also referred to in the art as the "Ion” gene
  • mutant capR phenotypes include: failure to lysogenize temperate phages [Takano, P.N.A.S. , 68, pp. 1469-1473 (1971)]; increased sensi ⁇ tivity to radiation [Howard-Flanders, et al., Genetics, 49, pp. 237-246 (1964) and Adler, et al. , J. Bacteriol. , 87, pp. 720-726 (1964)]; abnormal cell division [Walker, et al. , Mol,Gen.Genet. , 108, pp. 1296-1303 (1970) and Gayda, et al. , J. Bacteriol., 127, pp.
  • O PI involves derepression of the galETK operon and that the mucoid phenotype is not, by itself, responsible for other capR mutant phenotypes.
  • Gal " derivatives of capR mutants, for example, result in nonmucoid colonies that continue to exhibit the remaining capR mutant phenotypes. [See, e.g. - Markovitz (1977) supra and Gottesman, e_t al. , J. Bacteriol. , 133, pp. 844-851 (1978)] .
  • One particular dominant mucoid allele of the capR gene, called capR9 is of special significance to the present invention.
  • capR9 strains exhibit all of the standard phenotypes (mucoid appearance, radiation sensitivity, etc.) .
  • the mutant (mucoid) phenotype was dominant to the wild type (nonmucoid) phenotype. See, e.g. , Markovitz, et al. , P.N.A.S. , 4, pp.
  • capR9 When the capR9 was present in multiple copies (perhaps 2-3 times for an F' plasmid) , the defective subunits would dominate in the oligomer and the mutant phenotype would be expressed. This would be reversed when the wild type is in multiple copies. It is believed that the capR9 allele codes for a shortened polypeptide product because its phenotypes are partially reversed by ochre (nonesense) suppressors [Markovitz, et al. J.Bacteriol. , 94, pp. 338-395 (1967) and Markovitz (1977) supra.]
  • E__ coli strain CSR603 which displays the inability to repair damage to DNA from exposure to ultraviolet light.
  • the cells are transformed by plasmids which represent a smaller target for DNA damage and then irradiated with ultra ⁇ violet light, they are generally capable of expressing those proteins coded by the plasmid, but not by the chromosome.
  • Test cells were transformed with both plasmid pBZ201 containing the wild type capR gene and pBZ20lM9 which contained the capR9 mutation. The transformation procedure employed was as described in
  • 35 S-methionine was added to the culture medium con ⁇ taining the UV-treated cells , and .proteins harvested from the cells were thereafter subjected to electrophoretic analysis.
  • the control CSR603 cells showed no labelled proteins.
  • Cells transformed with the wild type plasmid displayed two distinct proteins, one of which is characteristic of the wild type capR gene coded by the plasmid.
  • CSR603 cells transformed with the capR9 plasmid displayed not only a labelled protein characteristic of the capR9 gene but also a wide array of unidentified cellular proteins not present in either the control GSR603 cells or those transformed with the pBZ201 plasmid.
  • Plasmid PEX2 is derived from plasmid pRI-11 which contains the rat proinsulin gene [Chan, et al. ,P.N.A.S. , 76, pp. 5036-5040 (1979)]. Plasmid PEX2 was engineered to contain the lac prompter DNA in ⁇ serted into the appropriate position of pRI-11 so that expression of synthesis of rat proinsulin messenger RNA and protein should occur. Radioimmunoassay [Rubenstein, et al. , Diabetes, 19, pp. 546-553 (1970)] for rat proinsulin product of cultured growth of the transformed cells detected no proinsulin product,
  • the "unsuccessful" transformant cells were thereafter further transformed with the capR9 plasmid, pBZ20lM9 (A.T.C.C.Plasmid 40041). Products of cells transformed with the capR9 allele were subjected to radioimmunoassay as above and revealed the presence of about 60 nanograms per liter of rat proinsulin which is the equivalent of approximately 100 molecules per cell.
  • capR capsular polysaccharide
  • gene such as capR9
  • Example 2 numerous alternatives to the Example 2 procedure of effecting a first transformation with a plasmid incorporating the exogenous gene and a second trans ⁇ formation with a vector incorporating the capR9 gene exist.
  • the mutant allele could be directly incorpor- ated into the chromosome.
  • a concurrent transformation of the E_ j _ coli host with both the selected exogenous gene and the capR9 gene could be made using a single hybrid vector incorporating both genes.
  • DNA plasmid pBZ20lM9 may itself be further hybridized, for example, to incorporate a selected exogenous gene coding for a protein of interest.
  • protein coded for by the dominant mucoid mutant gene functions to bind with and stabilize nucleic acids (specifically messenger RNA) in the host cell, preventing 'degradation and hence allowing for enhanced expression in the form of enhanced protein formation.
  • nucleic acids specifically messenger RNA
  • Such a theory provides a reasonable explanation for the results obtained in Example 1 above. More partic ⁇ ularly, the appearance of significant quantities of unidentified labelled protein in irradiated test cells could be due to the capR9 protein binding to m-RNA formed prior to irradiation (and chromosomal inacti- vation) . The m-RNA so stabilized and protected from degradation would be available to operate as a translational template for synthesis of protein incorporating the labelled methionine.
  • An alternative theory of operation of the invention has as its basis the ability of the protein coded for by the mutant allele to diminish activity of the wild type capR protein in degrading such proteins formed by the host cell which may be recognized in the cell as "foreign". This theory provides an explana ⁇ tion for the remarkable results obtained in Example 2 above.
  • capR9 shows no ATP-dependent protease activity, practically no ATPase activity in the presence of casein, and greatly reduced DNA-dependent ATPase activity.
  • I_n vitro experiments using mixtures of both capR and capR9 protein reveal significant diminution of wild type protein protease activity. Whether, in vivo, the mutant protein binds to (and represses activity of) the wild type protein or whether the putatively inactive mutant protein simply competes more effectively for binding sites than the wild type protein, the net result appears* to be enhanced expression of certain proteins in the form of diminished degradation thereof by the protease products of the wild type capR gene.
  • the overall result is enhanced expression of protein synthesis in terms of greater net quantities of isolatable products of exogenous and endogenous gene directed protein synthesis.
  • the present invention additionally compre ⁇ hends improvements in prior methods for securing production, in 1__ coli host cells, of protein coded for by exogenous genes, which improvements comprise employing E ⁇ _ coli host cells which contain a chor osomal mutation in the capsular polysaccharide, capR (Ion) , gene. While preferred methods involve use of E ⁇ _ coli cells having the above-noted dominant mucoid mutant allele (especially the capR9 allele) , also comprehended is the use of cells carrying other capR mutations, including recessive mutations. An example of the practice of this improved methodology is provided by the following description of work performed by collaborators Donald F.
  • Radioimmunoassays for rat proinsulin were carried out according to the procedures noted in Example 2. No rat proinsulin production was detected in the host cells of the first experiment. Rat proinsulin was detected in the host cells of the second experiment, wherein the host strain carried a mutation in the capR

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP19820902784 1981-08-21 1982-08-09 Expression der eiweisssynthese in e. coli. Withdrawn EP0093121A4 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US29512181A 1981-08-21 1981-08-21
US295121 1994-08-24

Publications (2)

Publication Number Publication Date
EP0093121A1 EP0093121A1 (de) 1983-11-09
EP0093121A4 true EP0093121A4 (de) 1984-02-09

Family

ID=23136297

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19820902784 Withdrawn EP0093121A4 (de) 1981-08-21 1982-08-09 Expression der eiweisssynthese in e. coli.

Country Status (3)

Country Link
EP (1) EP0093121A4 (de)
JP (1) JPS58501406A (de)
WO (1) WO1983000702A1 (de)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4758512A (en) * 1984-03-06 1988-07-19 President And Fellows Of Harvard College Hosts and methods for producing recombinant products in high yields
US4663281A (en) * 1984-03-22 1987-05-05 Mass Institute Of Technology Enhanced production of proteinaceous materials in eucaryotic cells
US5017477A (en) * 1985-10-25 1991-05-21 Biotechnica International, Inc. Enhancing DNA sequencies derived from the sacQ gene
AU582288B2 (en) * 1986-03-07 1989-03-16 Damon Biotech Inc. Vector and method for achieving high level expression in eukaryotic cells
US5143846A (en) * 1988-03-17 1992-09-01 The General Hospital Corporation Protease deficient bacterial hosts

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4329448A (en) * 1979-07-10 1982-05-11 Lever Brothers Company Microbial heteropolysaccharide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
No relevant documents have been disclosed. *
See also references of WO8300702A1 *

Also Published As

Publication number Publication date
JPS58501406A (ja) 1983-08-25
EP0093121A1 (de) 1983-11-09
WO1983000702A1 (en) 1983-03-03

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Inventor name: MARKOVITZ, ALVIN

Inventor name: CHARETTE, MARC F.

Inventor name: SCHOEMAKER, JOYCE M.

Inventor name: ZEHNBAUER, BARBARA A.