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WO1996016181A1 - Plasmide d'expression pour levure - Google Patents

Plasmide d'expression pour levure Download PDF

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Publication number
WO1996016181A1
WO1996016181A1 PCT/DE1995/001604 DE9501604W WO9616181A1 WO 1996016181 A1 WO1996016181 A1 WO 1996016181A1 DE 9501604 W DE9501604 W DE 9501604W WO 9616181 A1 WO9616181 A1 WO 9616181A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
expression plasmid
yeast
hpv
plasmid according
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.)
Ceased
Application number
PCT/DE1995/001604
Other languages
German (de)
English (en)
Inventor
Adrianns Johannes Cornelis Maria Braspenning
Wolfgang Z. Meschede
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.)
Deutsches Krebsforschungszentrum DKFZ
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
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 Deutsches Krebsforschungszentrum DKFZ filed Critical Deutsches Krebsforschungszentrum DKFZ
Priority to EP95937777A priority Critical patent/EP0792368A1/fr
Priority to JP8516442A priority patent/JPH10508754A/ja
Publication of WO1996016181A1 publication Critical patent/WO1996016181A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to yeast expression plasmids and yeast cells containing them.
  • the invention further relates to a method for producing polypeptides in yeast using the expression plasmids according to the invention.
  • yeast cells e.g. to use those of the Schizosaccharomyces Pombe strain for the expression of polypeptides.
  • the expressed polypeptides are usually not secreted into the medium, but accumulate in the yeast cells.
  • the yeast cells To isolate the polypeptides, the yeast cells must therefore be disrupted and the cell debris and yeast proteins obtained must be separated. This represents a time and financial expense.
  • the object of the present invention is therefore to provide an agent with which a polypeptide can be expressed in yeast cells without the above disadvantages occurring.
  • yeast expression plasmid which contains a DNA which codes for a signal peptide for yeast and a fusion polypeptide comprising polypeptide.
  • the polypeptide is expressed in the form of a fusion polypeptide in the yeast cells containing it.
  • the yeast signal peptide is cleaved off, as a result of which only the polypeptide is enriched in the medium.
  • Any vector suitable for expression in yeast can be used to produce a yeast expression plasmid according to the invention.
  • the specialist are such vectors are known.
  • pREP3-L20 This vector is derived from pREP3 (cf. Maundrell, K., Gene 123 (1993), pp. 127-130), but contains the following "multiple cloning site” (MCS):
  • a DNA is inserted into a vector above, which codes for a fusion polypeptide comprising a yeast signal peptide and a polypeptide.
  • the insertion takes place in such a way that the fusion polypeptide can be expressed in yeast cells. Suitable methods and conditions for this are known to the person skilled in the art. In particular, he makes sure that there is an ATG codon at the 5 'end of the DNA coding for the yeast signal peptide and for the polypeptide. It may also be advantageous if the 3 'end of the DNA coding for the yeast signal peptide is immediate, i.e. without via additional codons, adjoins the ATG codon of the DNA coding for the polypeptide.
  • yeast signal peptide refers to a peptide of any kind which enables a polypeptide to be transported outside in yeast.
  • this is a signal peptide derived from Saccharomyces Pombe.
  • Its DNA can have the sequence given in FIG. 1. It may be expedient to modify this sequence by one or more nucleotides, for example in the form of addition, deletion and / or substitution of nucleotides. Such a modification can, for example, aim at the 3 'end of the DNA coding for the yeast signal peptide being immediate borders on the ATG codon of the DNA coding for the polypeptide.
  • polypeptide refers to a polypeptide of any kind that can be expressed in yeast.
  • this is a non-yeast polypeptide.
  • proteins or parts thereof of human papilloma viruses HPV.
  • HPV human papilloma viruses
  • the fusion polypeptide encoded by an expression plasmid according to the invention comprises a signal peptide derived from Schizosaccharomyces Pombe, i.e. 1, and an E7 protein from HPV 16.
  • an expression plasmid contains the vector pREP3 L20 and is designated pREP3-L20 spl-HPV 16 E7. It was deposited with the DSM on October 28, 1994 under DSM 9532. The production of pREP3-L20 spl-HPV 16 E7 is shown in Fig. 2.
  • polypeptide indicates that such a product can also be the product of a fusion of two or more polypeptides.
  • polypeptides conveniently contain a region that can be easily bound by an antibody.
  • the fusion polypeptide encoded by an expression plasmid according to the invention comprises a signal peptide derived from Saccharomyces Pombe, ie one with the DNA sequence of FIG. 1, an E7 protein from HPV16 and a tag polypeptide.
  • the latter polypeptide comprises 1 1 amino acids of the C-terminus of SV40t antigen. It has a region that is bound by the commonly available antibody MAK tag (KT3).
  • An expression plasmid coding for the above fusion polypeptide contains the vector pREP3-L20 and is designated ⁇ REP3-L20 spl-HPV 16 E7 tag. It was deposited with the DSM on October 28, 1994 under DSM 9531.
  • the production of pREP3-L20 spl-HPV 16 E7 tag is shown in Fig. 3.
  • the expression plasmids according to the invention are distinguished in that the polypeptides expressed by the yeast cells are secreted into the medium.
  • the polypeptides can then be easily separated from the yeast cells by conventional methods such as centrifugation and subsequently purified. This is all the more true if the polypeptides contain a region that can easily be bound by an antibody.
  • the polypeptides can be captured via this region by an appropriate antibody, which is fixed, for example, to a column material.
  • the polypeptides are obtained in a purified form by subsequent elution.
  • the present invention also relates to yeast cells, preferably those of the Schizosaccharomyces Pombe strain, which contain an expression plasmid according to the invention, e.g. pREP3 L20 spl-HPV 16E7 or pREP3 L20 spl-HPV 16E7 tag included.
  • yeast cells preferably those of the Schizosaccharomyces Pombe strain, which contain an expression plasmid according to the invention, e.g. pREP3 L20 spl-HPV 16E7 or pREP3 L20 spl-HPV 16E7 tag included.
  • Another object of the present invention is a process for the production of polypeptides, which comprises the following process steps:
  • step (b) culturing the transformed yeast cells from step (a), thereby expressing a fusion polypeptide
  • FIG. 1 shows the DNA coding for a signal peptide from Schizosaccharomyces Pombe
  • FIG. 2 shows the expression plasmid pREP3-L20 spl-HPV 16E7 and its
  • Figure 3 shows the expression plasmid pREP3-L20 spl-HPV 16E7 tag and its preparation
  • FIG. 4 shows the DNA coding for the polypeptide tag and schematically the DNA coding for HPV 16E7 tag.
  • a single-stranded (ss) DNA coding for spl (cf. FIG. 1) is synthesized. This DNA is made double-stranded with the Klenow fragment (dsDNA spl) and then cut with the restriction enzymes BamHI and Nsil. A BamHI-Nsil DNA fragment is obtained.
  • the DNA coding for the protein E7 of HPV 16 (HPV 16E7) is amplified using a conventional PCR method.
  • the DNA obtained in this way is cut with the restriction enzymes Nsil and Sall.
  • An Nsil-SalI DNA fragment is obtained.
  • the above DNA fragments are used with the BamHI-SafI-cleaved vector Bluescript in a ligase reaction: the plasmid Bluescript spl HPV 16E7 is obtained. This plasmid is used with the restriction enzymes BamHI and Saf I cleaved and the DNA fragment containing spl and HPV 16E7 is isolated. This fragment is used together with the BamHI and Sai ⁇ cleaved vector pREP3-L20 (see above) in a ligase reaction. The expression plasmid pREP3-L20 spl-HPV 16E7 is obtained.
  • the plasmid Bluescript HPV 16E7 tag is produced analogously to the method described in Example 1. After cleavage with the restriction enzymes Nsil and Safl, the DNA fragment coding for HPV 16E7 tag (see FIG. 4) is isolated from this. This DNA fragment is inserted into the plasmid Bluescript spl HPV 16E7 (cf. Example 1) after it has been cleaved with Nsil and Safl and the DNA coding for HPV 16E7 has been removed. The plasmid Bluescript spl-HPV 16E7 day is obtained.
  • This plasmid is cleaved with the restriction enzymes Spei and Xhol and the spl-HPV 16E7 tag fragment is inserted into the correspondingly cleaved vector pREP3-L20 (see above).
  • the expression plasmid pREP3-L20 spl-HPV 16E7 day is obtained.
  • Transformed yeast cells are cultured, whereby the expression of the fusion polypeptide encoded by the expression plasmid takes place.
  • the yeast cells are centrifuged and the polypeptide HPV 16 E7 is detected in the cell supernatant.
  • Detection is carried out by dot blot analysis of the cell supernatant using the monoclonal antibody Mab HPV 16E7 IV directed against HPV 16E7.
  • intracellular and extracellular HPV 16E7 is detected in a Western blot.
  • the above-mentioned is used as the antibody.
  • polypeptide HPV 16E7 is secreted into the cell supernatant.
  • yeast cells of the strain Schizosaccharomyces Pombe LEU 1.32 are transformed with the expression plasmid pREP3-L20 spl-HPV 16E7 tag and the expression of the polypeptide HPV 16E7 tag is compared with the monoclonal antibody Mab tag () KT3) proven.
  • polypeptide HPV 16E7 is secreted into the cell supernatant.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un plasmide d'expression pour levure avec un ADN codant pour un polypeptide de fusion contenant un peptide signal pour levure et un polypeptide. En outre, l'invention concerne des cellules de levure renfermant un tel plasmide d'expression pour levure, ainsi qu'un procédé d'obtention de polypeptides avec utilisation dudit plasmide d'expression pour levure.
PCT/DE1995/001604 1994-11-18 1995-11-17 Plasmide d'expression pour levure Ceased WO1996016181A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP95937777A EP0792368A1 (fr) 1994-11-18 1995-11-17 Plasmide d'expression pour levure
JP8516442A JPH10508754A (ja) 1994-11-18 1995-11-17 酵母用発現プラスミド

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4441197.9 1994-11-18
DE19944441197 DE4441197C1 (de) 1994-11-18 1994-11-18 Expressionsplasmide für Hefe

Publications (1)

Publication Number Publication Date
WO1996016181A1 true WO1996016181A1 (fr) 1996-05-30

Family

ID=6533638

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE1995/001604 Ceased WO1996016181A1 (fr) 1994-11-18 1995-11-17 Plasmide d'expression pour levure

Country Status (4)

Country Link
EP (1) EP0792368A1 (fr)
JP (1) JPH10508754A (fr)
DE (1) DE4441197C1 (fr)
WO (1) WO1996016181A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002491A1 (fr) * 1995-07-04 1997-01-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procede pour deceler des anticorps specifiques diriges contre des proteines hpv
US7537009B2 (en) 2001-06-05 2009-05-26 Alexza Pharmaceuticals, Inc. Method of forming an aerosol for inhalation delivery

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9717953D0 (en) * 1997-08-22 1997-10-29 Smithkline Beecham Biolog Vaccine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0220689A2 (fr) * 1985-10-25 1987-05-06 Zymogenetics, Inc. Méthode permettant l'utilisation de BAR1 pour la sécrétion de protéines étrangères

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4005289A (en) * 1988-08-25 1990-03-01 Smithkline Beecham Corporation Recombinant saccharomyces

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0220689A2 (fr) * 1985-10-25 1987-05-06 Zymogenetics, Inc. Méthode permettant l'utilisation de BAR1 pour la sécrétion de protéines étrangères

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
B. CHAUDHURY ET AL.: "The pro-region of the yeast prepro-alfa-factor is essential for membrane translocation of human insulin-like growth factor 1 in vivo", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 206, pages 793 - 800 *
G. MATLASHEWSKI ET AL.: "The expression of human paillomavirus type 18 E6 protein in bacteria and the produiction of anti-E6 antibodies", JOURNAL OF GENERAL VIROLOGY, vol. 67, pages 1909 - 1916 *
H. PAN ET AL.: "Altered cell cycle regulation in the lens of HPV-16 E6 or E7 transgenic mice: implications for tumor suppressor gene function in development", GENES AND DEVELOPMENT, vol. 8, no. 1, pages 1285 - 1299 *
J. YANG ET AL.: "The structural gene coding for thiamin-repressible acid phosphatase in S. pombe", CURRENT GENETICS, vol. 18, pages 269 - 272 *
M. BIELEFELD ET AL.: "Bacterial beta-lactamase is efficiently secreted in S. cerevisiae under control of the invertase signal sequence", CURRENT GENETICS, vol. 21, pages 265 - 268 *
M. TOKUNAGA ET AL.: "Structure of yeast pGKL 128 kDa killer toxin secretion signal sequence", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 203, pages 415 - 423 *
S. ELLIOTT ET AL.: "Secretion of glycosylated huamn erytropoietin from yeast directed by the alfa-factor leader region", GENE, vol. 79, pages 167 - 180 *
S. ELLIOTT ET AL: "Isolation and characterization of the structuiral gene for secreted acid phosphatase from S. pombe", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 261, no. 6, pages 2936 - 2941 *
T. ACHSTETTER ET AL.: "A new signal peptide useful for secretion of heterologous proteins from yeast and its application for synthesis of hirudin", GENE, vol. 110, pages 25 - 31 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002491A1 (fr) * 1995-07-04 1997-01-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procede pour deceler des anticorps specifiques diriges contre des proteines hpv
US7537009B2 (en) 2001-06-05 2009-05-26 Alexza Pharmaceuticals, Inc. Method of forming an aerosol for inhalation delivery

Also Published As

Publication number Publication date
JPH10508754A (ja) 1998-09-02
DE4441197C1 (de) 1996-03-28
EP0792368A1 (fr) 1997-09-03

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