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EP0812362A1 - Plasmide pour l'expression de proteines de recombinaison modifiees dans un systeme bacterien - Google Patents

Plasmide pour l'expression de proteines de recombinaison modifiees dans un systeme bacterien

Info

Publication number
EP0812362A1
EP0812362A1 EP96906664A EP96906664A EP0812362A1 EP 0812362 A1 EP0812362 A1 EP 0812362A1 EP 96906664 A EP96906664 A EP 96906664A EP 96906664 A EP96906664 A EP 96906664A EP 0812362 A1 EP0812362 A1 EP 0812362A1
Authority
EP
European Patent Office
Prior art keywords
casein
human
plasmid
recombinant
protein
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Withdrawn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
EP96906664A
Other languages
German (de)
English (en)
Inventor
Pradip Mukerji
Jennifer Marie Thurmond
Lennart Hansson
Jeffrey Harris Baxter
Robert George Hards
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.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
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
Priority claimed from US08/554,137 external-priority patent/US5942254A/en
Priority claimed from US08/554,642 external-priority patent/US5710044A/en
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of EP0812362A1 publication Critical patent/EP0812362A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4732—Casein
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • human ⁇ —casein The primary structure of human ⁇ —casein was determined by Greenberg et al . (Journal of Biological Chemistry 259:5132-5138, 1984) . It was shown to be a phosphorylated protein with phosphorylation sites at specific seryl and threonyl residues located near the amino terminus. Comparison of human and bovine ⁇ - caseins showed 47% identity. The sequence of human
  • Hansson et al demonstrated that recombinant human ⁇ - casein was expressed in the yeast, S . cerevisiae, using the pYES 2.0 vector (Invitrogen Corp., San Diego, CA) . Production levels were estimated to be approximately 10% of the production found in E. coli .
  • recombinant ⁇ -casein obtained from S . cerevisiae a eukaryotic cell that has endogenous enzymes capable of phosphorylating proteins, was phosphorylated, but the protein produced by E. coli , a prokaryotic cell that lacks the ability in its native state to phosphorylate, was non-phosphorylated.
  • recombinant human casein kinase II (rhCKII) produced in and purified from E. coli can phosphorylate protein substrates in vi tro (Shi et al. , Proceeding of the National Academy of Sciences, USA 91:2767-2771, 1994) .
  • One specific embodiment of the present invention uses a nucleotide sequence encoding a recombinant human casein kinase II in a single construct with nucleotide sequence encoding ⁇ -casein to transform E. coli and produce phosphorylated ⁇ -casein.
  • exogenous proteins capable of being modified through the process of the present invention include but are not limited to human caseins, including ⁇ -casein, cell receptor proteins, fatty acylated proteins including palmitoylated proteins, mammalian muscle proteins, the gag polyproteins of retroviruses, and mammalian proteins targeted by retroviral src kinases.
  • Transmembrane glycoproteins that acquire covalent palmitate after synthesis include the insulin, ⁇ 2 -adrenergic and transferrin receptors.
  • Figure 1 shows physical maps of expression vectors pS637 and pRJB-6 constructed for inducible intracellular expression in E. coli . 191 base pairs were removed from pS637 to produce PRJB-6.
  • lane 2 native human ⁇ -casein
  • lane 3 induced HMS174 (DE3 )pLysS (pRJB-9)
  • lane 4 induced HMS174 (DE3 )pLysS (pS637)
  • lane 5 induced HMS174 (DE3 )pLysS (pS637)
  • Nucleotide sequences encoding ⁇ —casein in several different expression formats, were evaluated for expression of recombinant human ⁇ -casein in an E. coli strain. After a series of experiments, it was determined that recombinant human ⁇ -casein was efficiently phosphorylated when sequences encoding human ⁇ —casein were placed in a single construct with sequences encoding human casein kinase CKII ⁇ . Efficiency of phosphorylation was not compromised when both genes were placed in tandem in one plasmid when compared with experimental systems in which sequences encoding the kinase and the ⁇ -casein were placed in two separate vectors.
  • Electrophoresis and Detection of Recombinant ⁇ -Casein Cells were pelleted by centrifugation and the pellet from 1 ml of culture was dissolved in 100 ⁇ l of sample buffer, which contains Tris, glycerol, SDS, dithiothreotol (DTT) , and bromophenol blue.
  • sample buffer which contains Tris, glycerol, SDS, dithiothreotol (DTT) , and bromophenol blue.
  • the proteins were separated by SDS-PAGE as described in Laemmli (Nature 227:680-685, 1970) .
  • Gradient gels were cast and run in the discontinuous buffer system in a Protean (Bio-Rad, Richmond, CA) electrophoresis unit. Gels were stained as described in Laemmli. Immunoblotting was performed according to the specifications of the manufacturer (Bio-Rad) .
  • the Western blot shown in Figure 8 shows reduced production of recombinant CKII ⁇ -casein by induced cells containing pRJB-7 when compared with cells containing pRJB-9. This is seen by comparing lane 4 (induced pRJB-7) with lane 8 (induced pRJB-9) . Although both pRJB-7 and pRJB-9 are derived from pS637, only pRJB-9 produced amounts of CKII ⁇ -casein equivalent to the parent construct. The presence of an additional T7 promoter before the CKII genes in the hybrid construct had the effect of both reducing cell growth and consequently reducing recombinant protein production.
  • Figure 9 shows a Western blot analysis in which the lysates were developed with phosphoserine antibody to detect phosphorylated protein. Induced E. coli
  • HMS174 (DE3)LysS cells containing pET-lld- CKII ⁇ , pRJB-9 (hybrid construct with one T7 promoter) , pRJB-7 (hybrid construct with two T7 promoters) , or pS637 (contains ⁇ -casein encoding sequence but not CKII ⁇ encoding sequence) were compared for production of phosphorylated recombinant ⁇ -casein. Phosphorylated ⁇ - casein was produced only in cells containing pRJB-9 (lane 6) . No phosphorylated protein was detected in lane 7, which contains the lysate of cells containing pRJB-7.
  • Example 4 Production of human ⁇ -casein in E. coli K-12: Construct pRJB-9 containing both ⁇ -casein encoding sequence and CKII ⁇ encoding sequences
  • the present invention uses a single construct expressing both the information for transferring functional groups to specific sites and the protein to be modified.
  • the transferred functional group is phosphate.
  • the transfer is accomplished by a kinase that is demonstrated to mediate phosphorylation of specific sites on recombinant human ⁇ -casein in vivo .
  • This invention demonstrates that not only can human ⁇ —casein be specifically phosphorylated in vivo by E. coli , but that a single-construct with a promoter located before the sequence encoding ⁇ -casein and having the advantages of a single-construct system can successfully mediate this function.
  • H. influenzae has been identified as a causative factor for otitis media (Murray et al . , 1994) . Since it has been demonstrated in the experiments described above that recombinant human ⁇ -casein phosphorylated in at least three sites under the direction of the plasmid of the invention inhibits adhesion of H. influenzae to human cells, it is concluded that phosphorylated recombinant human ⁇ -casein, as described above, may be used in the prevention and treatment of otitis media in humans, particularly in human infants.
  • This invention will allow commercial-scale production of phosphorylated, recombinant mammalian proteins in microorganisms.
  • the method of the invention can be used to produce recombinant exogenous proteins, including but not limited to, recombinant human ⁇ — casein, in large quantities.
  • Phosphorylation of ⁇ - casein in a bioreactor makes possible large-scale synthesis in a fermentor of recombinant ⁇ -casein that is equivalent to native human ⁇ -casein. This will facilitate the production of infant formula containing human ⁇ -casein in its native phosphorylated state.
  • the method of the invention can also be used for phosphorylation of cell proteins, including receptors which are regulated by phosphorylation and dephosphorylation and thereby act as signals in cell metabolism.
  • the invention provides a cost-effective method of phosphorylating peptide receptors and will be useful in the manufacture of pharmaceutical drugs.
  • the single plasmid system is preferable to a two-plasmid system for industrial production of fermented proteins such as recombinant, phosphorylated human ⁇ —casein.
  • MOLECULE TYPE genomic DNA

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention décrit un plasmide contenant une séquence de promoteurs, une séquence nucléotidique codant une protéine exogène et une séquence nucléotidique codant un enzyme capable de modifier cette protéine exogène. Dans un mode de réalisation spécifique, la protéine exogène codée est la caséine β humaine et l'enzyme codé est une kinase humaine capable de réaliser la phosphorylation de la caséine β de recombinaison dans un système bactérien.
EP96906664A 1995-02-27 1996-02-27 Plasmide pour l'expression de proteines de recombinaison modifiees dans un systeme bacterien Withdrawn EP0812362A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US39523995A 1995-02-27 1995-02-27
US395239 1995-02-27
US08/554,137 US5942254A (en) 1995-02-27 1995-11-06 Phosphorylated recombinant human β-casein expressed in a bacterial system
US08/554,642 US5710044A (en) 1995-02-27 1995-11-06 Plasmid for expressing phosphorylated recombinant proteins in a bacterial system
US554137 1995-11-06
US554642 1995-11-06
PCT/US1996/002866 WO1996027018A1 (fr) 1995-02-27 1996-02-27 Plasmide pour l'expression de proteines de recombinaison modifiees dans un systeme bacterien

Publications (1)

Publication Number Publication Date
EP0812362A1 true EP0812362A1 (fr) 1997-12-17

Family

ID=27410154

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96906664A Withdrawn EP0812362A1 (fr) 1995-02-27 1996-02-27 Plasmide pour l'expression de proteines de recombinaison modifiees dans un systeme bacterien

Country Status (5)

Country Link
EP (1) EP0812362A1 (fr)
JP (1) JPH11500920A (fr)
CA (1) CA2213734A1 (fr)
NZ (1) NZ303621A (fr)
WO (1) WO1996027018A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5707968A (en) * 1994-05-26 1998-01-13 Abbott Laboratories Inhibition of attachment of H.influenzae to human cells
US5942254A (en) * 1995-02-27 1999-08-24 Abbott Laboratories Phosphorylated recombinant human β-casein expressed in a bacterial system
WO1997017085A2 (fr) * 1995-11-06 1997-05-15 Abbott Laboratories Procede permettant d'inhiber la fixation de h. influenzae sur des cellules humaines a l'aide de beta-caseine humaine phosphorylee de recombinaison
JP2000503319A (ja) * 1995-11-06 2000-03-21 アボツト・ラボラトリーズ インフルエンザ菌のヒト細胞への付着を防止するための生成物
US6287866B1 (en) * 1996-11-27 2001-09-11 Abbott Laboratories β-casein expressing constructs

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0178863A1 (fr) * 1984-10-15 1986-04-23 Schering Corporation Systèmes d'expression utilisant des promoteurs de bactériophage T7 et des séquences de gènes
DK0445097T3 (da) * 1990-02-26 1996-09-16 Univ Washington Fremgangsmåde til N-myristoylering af protein
IE914102A1 (en) * 1990-11-26 1992-06-03 Genetics Inst Expression of pace in host cells and methods of use thereof
EP0548012B1 (fr) * 1991-12-16 1997-09-03 Novartis AG Endoprotéase dibasique recombinante localisée dans le réticulum endoplasmique et ses applications
US5512434A (en) * 1992-12-14 1996-04-30 The United States Of America As Represented By The Department Of Health And Human Services Expression cloning of a human phosphatase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL: "costimulationof antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4", CELL, vol. 71, 24 December 1992 (1992-12-24), pages 1093 - 1102 *

Also Published As

Publication number Publication date
NZ303621A (en) 1999-04-29
JPH11500920A (ja) 1999-01-26
CA2213734A1 (fr) 1996-09-06
WO1996027018A1 (fr) 1996-09-06

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