[go: up one dir, main page]

WO1996016181A1 - Expression plasmids for yeasts - Google Patents

Expression plasmids for yeasts Download PDF

Info

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)
French (fr)
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 JP8516442A priority Critical patent/JPH10508754A/en
Priority to EP95937777A priority patent/EP0792368A1/en
Publication of WO1996016181A1 publication Critical patent/WO1996016181A1/en
Anticipated expiration legal-status Critical
Ceased 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
    • C07K14/005—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.

Landscapes

  • 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

The invention relates to a yeast expression plasmid with a DNA which codes a fusion polypeptide containing a signal peptide for yeast and a polypeptide. The invention also relates to yeast cells containing such a yeast expression plasmid and a process for producing polypeptides using the yeast expression plasmid.

Description

Expressionsplasmide für HefeExpression plasmids for yeast

Die Erfindung betrifft Hefe-Expressionsplasmide und solche enthaltende Hefezellen. Ferner betrifft die Erfindung ein Verfahren zur Herstellung von Polypeptiden in Hefe unter Verwendung der erfindungsgemäßen Expressionsplasmide.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.

Es ist bekannt, Hefezellen, z.B. solche des Stammes Schizosaccharomyces Pombe, zur Expression von Polypeptiden zu verwenden. Die exprimierten Polypeptide werden aber in der Regel nicht in das Medium sekretiert, sondern lagern sich in den Hefezellen an. Zur Isolierung der Polypeptide müssen daher die Hefezellen aufgeschlossen und die erhaltenen Zelltrümmer und Hefeproteine abgetrennt werden. Dies stellt einen zeitlich und finanziell großen Aufwand dar.It is known to use yeast cells e.g. to use those of the Schizosaccharomyces Pombe strain for the expression of polypeptides. However, the expressed polypeptides are usually not secreted into the medium, but accumulate in 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.

Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzu¬ stellen, mit dem ein Polypeptid in Hefezellen exprimiert werden kann, ohne daß vorstehende Nachteile auftreten.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.

Erfindungsgemäß wird dies durch ein Hefe-Expressionsplasmid erreicht, das eine DNA enthält, die für ein Signalpeptid für Hefe und ein Polypeptid umfassendes Fu¬ sionspolypeptid codiert.According to the invention, this is achieved by a yeast expression plasmid which contains a DNA which codes for a signal peptide for yeast and a fusion polypeptide comprising polypeptide.

Mit einem solchen Hefe-Expressionsplasmid wird in dieses enthaltenden Hefezellen das Polypeptid in Form eines Fusionspolypeptids exprimiert. Dieses enthält ein Hefe-Signalpeptid, wodurch das Fusionspolypeptid aus den Hefezellen heraustrans¬ portiert wird. Hierbei wird das Hefe-Signalpeptid abgespalten, wodurch im Medium nur das Polypeptid angereichert wird.With such a yeast expression plasmid, the polypeptide is expressed in the form of a fusion polypeptide in the yeast cells containing it. This contains a yeast signal peptide, as a result of which the fusion polypeptide is transported out of the yeast cells. The yeast signal peptide is cleaved off, as a result of which only the polypeptide is enriched in the medium.

Zur Herstellung eines erfindungsgemäßen Hefe-Expressionsplasmids kann jeglicher zur Expression in Hefe geeignete Vektor verwendet werden. Dem Fachmann sind solche Vektoren bekannt. Beispielsweise wird er auf pREP3-L20 zurückgreifen. Dieser Vektor leitet sich von pREP3 (vgl. Maundrell, K., Gene 123 (1993), S. 127- 130) ab, enthält aber nachstehende "Multiple Cloning Site" (MCS):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. For example, he will use 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):

L-20 Mscl BamHI Spei Notl Sall Apal Xhol Smal aaattggCCAAGGATCCAACTAGTAGCGGCCGCAGTCGACAGGGCCCACTCGAGACCCgggt tttaaccGGTTCCTAGGTTGATCATCGCCGGCGTCAGCTGTCCCGGGTGAGCTCTGGGcccaL-20 Mscl BamHI Spei Notl Sall Apal Xhol Smal aaattggCCAAGGATCCAACTAGTAGCGGCCGCAGTCGACAGGGCCCACTCGAGACCCgggt tttaaccGGTTCCTAGGTTGATCATCGCCGGCGTCAGCTGTCCCGTGGGGcaTCAGCTGTCCCGTGGGGcaC

In einen vorstehenden Vektor wird eine DNA inseriert, die für ein ein Hefe-Signal¬ peptid und ein Polypeptid umfassendes Fusionspolypeptid codiert. Die Insertion erfolgt derart, daß das Fusionspolypeptid in Hefezellen exprimiert werden kann. Dem Fachmann sind hierfür geeignete Verfahren und Bedingungen bekannt. Insbesondere achtet er darauf, daß jeweils am 5'-Ende der für das Hefe-Signalpep¬ tid und der für das Polypeptid codierenden DNA ein ATG-Codon vorliegt. Auch kann es von Vorteil sein, wenn das 3'-Ende der für das Hefe-Signalpeptid codieren¬ den DNA unmittelbar, d.h. ohne über zusätzliche Codons, an das ATG-Codon der für das Polypeptid codierenden DNA grenzt. Günstig kann es ferner sein, die für das Hefe-Signalpeptid codierende DNA zunächst mit der für das Polypeptid codie¬ renden DNA zu verknüpfen und dann das Ligationsprodukt in den Vektor zu inserieren. Andererseits liefert die getrennte Insertion der beiden DNAs, d.h. wenn nur die für das Hefe-Signalpeptid codierende DNA, inseriert wird, einen neuen vorteilhaften Expressionsvektor für Hefen. Ein solcher Vektor ist ebenfalls Gegen¬ stand der vorliegenden Erfindung.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. It may also be advantageous to first link the DNA coding for the yeast signal peptide to the DNA coding for the polypeptide and then insert the ligation product into the vector. On the other hand, the separate insertion of the two DNAs, i.e. if only the DNA coding for the yeast signal peptide is inserted, a new advantageous expression vector for yeast. Such a vector is also the subject of the present invention.

Der vorstehende Ausdruck "Hefe-Signalpeptid" betrifft ein Peptid jeglicher Art, das in Hefen den Transport eines Polypeptids nach außen ermöglicht. Dies ist z.B. ein aus Saccharomyces Pombe stammendes Signalpeptid. Seine DNA kann die in Fig. 1 angegebene Sequenz aufweisen. Günstig kann es sein, diese Sequenz durch ein oder mehrere Nukleotide, z.B. in Form von Addition, Deletion und/oder Substitution von Nukleotiden, zu modifizieren. Eine solche Modifikation kann z.B. darauf ab¬ zielen, daß das 3'-Ende der für das Hefe-Signalpeptid codierenden DNA unmittelbar an das ATG-Codon der für das Polypeptid codierenden DNA grenzt.The above expression "yeast signal peptide" refers to a peptide of any kind which enables a polypeptide to be transported outside in yeast. For example, 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.

Ferner betrifft der vorstehende Ausdruck "Polypeptid" ein Polypeptid jeglicher Art, das in Hefen exprimiert werden kann. Vorzugsweise ist dies ein nicht von Hefen stammendes Polypeptid. Beispiele hierfür sind Proteine oder Teile davon von hu¬ manen Papillomviren (HPV). Von diesen sind die frühen, ein Kernsignal aufweisen¬ den Proteine E6 und E7 von HPV 11, HPV 16 und HPV 18 besonders zu erwäh¬ nen.Furthermore, the above term "polypeptide" refers to a polypeptide of any kind that can be expressed in yeast. Preferably, this is a non-yeast polypeptide. Examples of this are proteins or parts thereof of human papilloma viruses (HPV). Of these, the early proteins E6 and E7 of HPV 11, HPV 16 and HPV 18, which have a core signal, are particularly worth mentioning.

In bevorzugter Ausführungsform umfaßt das durch ein erfindungsgemäßes Ex- pressionsplasmid codierte Fusionspolypeptid ein von Schizosaccharomyces Pombe stammendes Signalpeptid, d.h. eines mit der DNA-Sequenz von Fig. 1 , und ein E7- Protein von HPV 16. Ein solches Expressionsplasmid enthält den Vektor pREP3 L20 und wird mit pREP3-L20 spl-HPV 16 E7 bezeichnet. Es wurde bei der DSM am 28. 10. 1994 unter DSM 9532 hinterlegt. Die Herstellung von pREP3-L20 spl- HPV 16 E7 wird in Fig. 2 gezeigt.In a preferred embodiment, 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. Such 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.

Desweiteren weist der Ausdruck "Polypeptid" darauf hin, daß ein solches auch das Produkt einer Fusion aus zwei oder mehreren Polypeptiden sein kann. Eines oder mehrere dieser Polypeptide enthalten günstigerweise eine Region, die leicht von einem Antikörper gebunden werden kann.Furthermore, the term "polypeptide" indicates that such a product can also be the product of a fusion of two or more polypeptides. One or more of these polypeptides conveniently contain a region that can be easily bound by an antibody.

In bevorzugter Ausführungsform umfaßt das durch ein erfindungsgemäßes Ex¬ pressionsplasmid codierte Fusionspolypeptid ein von Saccharomyces Pombe stammendes Signalpeptid, d.h. eines mit der DNA-Sequenz von Fig. 1 , ein E7- Protein von HPV16 und ein tag Polypeptid. Letzteres Polypeptid umfaßt 1 1 Amino¬ säuren des C-Terminus von SV40t-Antigen. Es weist eine Region auf, die von dem allgemein erhältlichen Antikörper MAK tag (KT3) gebunden wird. Ein für vorstehen¬ des Fusionspolypeptid codierendes Expressionsplasmid enthält den Vektor pREP3- L20 und wird mit ρREP3-L20 spl-HPV 16 E7 tag bezeichnet. Es wurde bei der DSM am 28. 10. 1994 unter DSM 9531 hinterlegt. Die Herstellung von pREP3- L20 spl-HPV 16 E7 tag wird in Fig. 3 gezeigt. Die erfindungsgemäßen Expressionsplasmide zeichnen sich dadurch aus, daß die von den Hefezellen exprimierten Polypeptide in das Medium sekretiert werden. Die Polypeptide können dann leicht von den Hefezellen durch übliche Verfahren, wie Zentrifugation, getrennt und nachfolgend gereinigt werden. Dies gilt umso mehr, wenn die Polypeptide eine Region enthalten, die leicht von einem Antikörper gebunden werden kann. Über diese Region können die Polypeptide durch einen entsprechenden Antikörper gefangen werden, der z.B. an einem Säulenmaterial fixiert ist. Durch nachfolgende Elution werden die Polypeptide in gereinigter Form erhalten.In a preferred embodiment, 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 ρREP3-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.

Gegenstand der vorliegenden Erfindung sind auch Hefezellen, vorzugsweise solche des Stammes Schizosaccharomyces Pombe, die ein erfindungsgemäßes Expres¬ sionsplasmid, z.B. pREP3 L20 spl-HPV 16E7 oder pREP3 L20 spl-HPV 16E7 tag enthalten.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.

Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Her¬ stellung von Polypeptiden, das folgende Verfahrensschritte umfaßt:Another object of the present invention is a process for the production of polypeptides, which comprises the following process steps:

(a) Transformation von Hefezellen, insbesondere des Stammes Schizosaccharo¬ myces Pombe, mit einem vorstehenden Expressionsplasmid,(a) transformation of yeast cells, in particular of the Schizosaccharo¬ myces Pombe strain, with an above expression plasmid,

(b) Kultivierung der transformierten Hefezellen von Schritt (a), wodurch ein Fusionspolypeptid exprimiert wird, und(b) culturing the transformed yeast cells from step (a), thereby expressing a fusion polypeptide, and

(c) Abtrennung des im Zellüberstand vorliegenden Polγpeptids und gegebenen¬ falls weitere Reinigung des Polypeptids.(c) separation of the polypeptide present in the cell supernatant and, if appropriate, further purification of the polypeptide.

Zur Durchführung vorstehender Verfahren sind dem Fachmann geeignete Materia¬ lien und Bedingungen bekannt. Ergänzend wird auf Maniatis et al., Molecular Cloning: A laboratory mannual (1982), Cold Spring Harbor, New York, verwiesen. Kurze Beschreibung der Zeichnung:Suitable materials and conditions are known to the person skilled in the art for carrying out the above processes. In addition, reference is made to Maniatis et al., Molecular Cloning: A laboratory mannual (1982), Cold Spring Harbor, New York. Brief description of the drawing:

Figur 1 zeigt die für ein Signalpeptid von Schizosaccharomyces Pombe codie¬ rende DNA,FIG. 1 shows the DNA coding for a signal peptide from Schizosaccharomyces Pombe,

Figur 2 zeigt das Expressionsplasmid pREP3-L20 spl-HPV 16E7 sowie seineFIG. 2 shows the expression plasmid pREP3-L20 spl-HPV 16E7 and its

Herstellung,Manufacture,

Figur 3 zeigt das Expressionsplasmid pREP3-L20 spl-HPV 16E7 tag sowie seine Herstellung, undFigure 3 shows the expression plasmid pREP3-L20 spl-HPV 16E7 tag and its preparation, and

Figur 4 zeigt die für das Polypeptid tag codierende DNA sowie schematisch die für HPV 16E7 tag codierende DNA.FIG. 4 shows the DNA coding for the polypeptide tag and schematically the DNA coding for HPV 16E7 tag.

Die folgenden Beispiele erläutern die Erfindung.The following examples illustrate the invention.

Beispiel 1example 1

Herstellung des Expressionsplasmids pREP3-L20 spl-HPV 16E7Preparation of the expression plasmid pREP3-L20 spl-HPV 16E7

Die Herstellung dieses Expressionsplasmids ist schematisch in Figur 2 dargestellt.The production of this expression plasmid is shown schematically in FIG. 2.

Es wird eine einzelsträngige (ss) für spl (vgl. Fig. 1 ) codierende DNA synthetisiert. Mit dem Klenow-Fragment wird diese DNA doppelsträngig gemacht (dsDNA spl) und anschließend mit den Restriktionsenzymen BamHI und Nsil nachgeschnitten. Es wird ein BamHI-Nsil DNA-Fragment erhalten.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.

Ferner wird mit einem üblichen PCR- Verfahren die für das Protein E7 von HPV 16 codierende DNA (HPV 16E7) amplifiziert. Die so erhaltene DNA wird mit den Restriktiόnsenzymen Nsil und Sall nachgeschnitten. Es wird ein Nsil-Sall DNA- Fragment erhalten.Furthermore, 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.

Vorstehende DNA-Fragmente werden mit dem BamHI-SafI gespaltenen Vektor Bluescript in eine Ligasereaktion eingesetzt: Es wird das Plasmid Bluescript spl HPV 16E7 erhalten. Dieses Plasmid wird mit den Restriktionsenzymen BamHI und Saf I gespalten und das spl und HPV 16E7 enthaltende DNA-Fragment wird isoliert. Dieses Fragment wird zusammen mit dem BamHI und Sai\ gespaltenen Vektor pREP3-L20 (vgl. vorstehend) in eine Ligasereaktion eingesetzt. Es wird das Ex¬ pressionsplasmid pREP3-L20 spl-HPV 16E7 erhalten.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.

Beispiel 2Example 2

Herstellung des Expressionsplasmids pREP3-L20 spl-HPV 16E7 tagProduction of the expression plasmid pREP3-L20 spl-HPV 16E7 tag

Die Herstellung dieses Expressionsplasmids ist schematisch in Figur 3 dargestellt.The production of this expression plasmid is shown schematically in FIG. 3.

Analog zu dem in Beispiel 1 beschriebenen Verfahren wird das Plasmid Bluescript HPV 16E7 tag hergestellt. Aus diesem wird nach Spaltung mit den Restriktions¬ enzymen Nsil und Safl das für HPV 16E7 tag codierende DNA-Fragment (vgl. Figur 4) isoliert. Dieses DNA-Fragment wird in das Plasmid Bluescript spl HPV 16E7 (vgl. Beispiel 1 ) inseriert, nachdem dieses mit Nsil und Safl gespalten und die für HPV 16E7-codierenden DNA entfernt worden sind. Es wird das Plasmid Bluescript spl-HPV 16E7 tag erhalten. Dieses Plasmid wird mit den Restriktions¬ enzymen Spei und Xhol gespalten und das spl-HPV 16E7 tag-Fragment wird in den entsprechend gespaltenen Vektor pREP3-L20 (vgl. vorstehend) inseriert. Es wird das Expressionsplasmid pREP3-L20 spl-HPV 16E7 tag erhalten.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.

Beispiel 3Example 3

Expression des Proteins E7 von HPV 16 (HPV 16E7)Expression of protein E7 from HPV 16 (HPV 16E7)

(a) Das Expressionsplasmid pREP3-L20 spl HPV-16E7 wird zur Transformation von Hefezellen des Stammes Schizosaccharomyces Pombe LEU 1.32 ver¬ wendet. Diese Hefezellen können kein Leucin herstellen. Dieser Mangel wird aber durch vorstehendes Expressionsplasmid überwunden, da dieses auch für Leucin codiert. Es findet eine Selektion in einem Leucin-Mangelmedium statt.(a) The expression plasmid pREP3-L20 spl HPV-16E7 is used for the transformation of yeast cells of the Schizosaccharomyces Pombe LEU 1.32 strain. These yeast cells cannot produce leucine. However, this deficiency is overcome by the above expression plasmid, since this also codes for leucine. A selection is made in a leucine deficient medium instead of.

Transformierte Hefezellen werden kultiviert, wodurch die Expression des durch das Expressionsplasmid codierten Fusionspolypeptids stattfindet. Die Hefezellen werden zentrifugiert und das Polypeptid HPV 16 E7 wird im Zellüberstand nachgewiesen.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.

Der Nachweis erfolgt durch Dot-blot-Analγse des Zellüberstands mittels des monoklonalen gegen HPV 16E7 gerichteten Antikörpers Mab HPV 16E7 IV.Detection is carried out by dot blot analysis of the cell supernatant using the monoclonal antibody Mab HPV 16E7 IV directed against HPV 16E7.

Ferner findet ein Nachweis von intra- und extrazellulärem HPV 16E7 in einem Western-Blot statt. Als Antikörper wird vorstehend genannter ver¬ wendet.In addition, intracellular and extracellular HPV 16E7 is detected in a Western blot. The above-mentioned is used as the antibody.

Es zeigt sich, daß das Polypeptid HPV 16E7 in den Zeilüberstand sekretiert wird.It is shown that the polypeptide HPV 16E7 is secreted into the cell supernatant.

(b) Analog zu vorstehendem Verfahren von (a) werden Hefezellen des Stammes Schizosaccharomyces Pombe LEU 1.32 mit dem Expressionsplasmid pREP3- L20 spl-HPV 16E7 tag transformiert und die Expression des Polypeptids HPV 16E7 tag wird mit dem gegen tag gerichteten monoklonalen Antikörper Mab tag (KT3) nachgewiesen.(b) Analogous to the above method of (a), 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.

Es zeigt sich, daß das Polypeptid HPV 16E7 tag in den Zellüberstand sekre¬ tiert wird.It can be seen that the polypeptide HPV 16E7 is secreted into the cell supernatant.

Die in den Beispielen beschriebenen Verfahren und Materialien sind dem Fachmann bekannt. Ergänzend wird auf Maniatis et al., vorstehend angege¬ ben, verwiesen. The processes and materials described in the examples are known to the person skilled in the art. In addition, reference is made to Maniatis et al., Given above.

Claims

Patentansprüche claims 1. Hefe-Expressionsplasmid mit einer DNA, die für ein ein Signalpeptid für Hefe und ein Polypeptid umfassendes Fusionspolypeptid codiert.1. Yeast expression plasmid with a DNA coding for a fusion polypeptide comprising a signal peptide for yeast and a polypeptide. 2. Hefe-Expressionsplasmid nach Anspruch 1 , dadurch gekennzeichnet, daß das Signalpeptid von Schizosaccharomyces Pombe stammt.2. Yeast expression plasmid according to claim 1, characterized in that the signal peptide is derived from Schizosaccharomyces Pombe. 3. Hefe-Expressionsplasmid nach Anspruch 2, dadurch gekennzeichent, daß das Signalpeptid jenes von Fig. 1 ist.3. yeast expression plasmid according to claim 2, characterized in that the signal peptide is that of Fig. 1. 4. Hefe-Expressionsplasmid nach einem der Ansprüche 1 - 3, dadurch gekenn¬ zeichnet, daß das Polypeptid nicht von einer Hefe stammt.4. yeast expression plasmid according to any one of claims 1-3, characterized gekenn¬ characterized in that the polypeptide does not come from a yeast. 5. Hefe-Expressionsplasmid nach einem der Ansprüche 1 - 3, dadurch gekenn¬ zeichnet, daß das Polypeptid von einem humanpathogenen Papillomvirus (HPV) stammt.5. yeast expression plasmid according to any one of claims 1-3, characterized gekenn¬ characterized in that the polypeptide is derived from a human pathogenic papilloma virus (HPV). 6. Hefe-Expressionsplasmid nach Anspruch 5, dadurch gekennzeichnet, daß das Polypeptid ein HPV E6- oder HPV E7-Protein ist.6. yeast expression plasmid according to claim 5, characterized in that the polypeptide is an HPV E6 or HPV E7 protein. 7. Hefe-Expressionsplasmid nach Anspruch 6, dadurch gekennzeichnet, daß das E6- und E7-Protein von HPV 1 1 , HPV 16 und HPV 18 stammt.7. yeast expression plasmid according to claim 6, characterized in that the E6 and E7 protein is derived from HPV 1 1, HPV 16 and HPV 18. 8. Hefe-Expressionsplasmid nach Anspruch 7, hinterlegt bei der DSM unter DSM 9532.8. yeast expression plasmid according to claim 7, deposited with the DSM under DSM 9532. 9. Hefe-Expressionsplasmid nach einem der Ansprüche 1 - 7, dadurch gekenn¬ zeichnet, daß das Polypeptid mit einem weiteren Polypeptid fusioniert ist. 9. yeast expression plasmid according to any one of claims 1-7, characterized gekenn¬ characterized in that the polypeptide is fused with another polypeptide. 10. Hefe-Expressionsplasmid nach Anspruch 9, dadurch gekennzeichent, daß das weitere Polypeptid eine Antikörper-Bindungsregion umfaßt.10. Yeast expression plasmid according to claim 9, characterized in that the further polypeptide comprises an antibody binding region. 1 1. Expressionsplasmid nach Anspruch 10, hinterlegt bei der DSM unter DSM 9531.1 1. Expression plasmid according to claim 10, deposited with the DSM under DSM 9531. 12. Hefezellen, enthaltend den Vektor nach einem der Ansprüche 1 - 1 1 .12. yeast cells containing the vector according to any one of claims 1 - 1 1. 13. Hefezellen nach Anspruch 12, dadurch gekennzeichnet, daß es solche des Stammes Schizosaccharomyces Pombe sind.13. Yeast cells according to claim 12, characterized in that they are those of the Schizosaccharomyces Pombe strain. 14. Verfahren zur Herstellung von Polypeptiden, umfassend die folgenden Verfahrensschritte:14. A process for the preparation of polypeptides, comprising the following process steps: (a) Transformation von Hefezellen mit dem Expressionsplasmid nach einem der Ansprüche 1 - 1 1 ,(a) transformation of yeast cells with the expression plasmid according to any one of claims 1 - 1 1, (b) Kultivierung der transformierten Hefezellen von Schritt (a), wodurch das Fusionspolypeptid exprimiert wird, und(b) culturing the transformed yeast cells from step (a), thereby expressing the fusion polypeptide, and (c) Abtrennung des im Zellüberstand vorliegenden Polypeptids und ggfs. weitere Reinigung des Polypeptids. (c) separation of the polypeptide present in the cell supernatant and, if necessary, further purification of the polypeptide.
PCT/DE1995/001604 1994-11-18 1995-11-17 Expression plasmids for yeasts Ceased WO1996016181A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP8516442A JPH10508754A (en) 1994-11-18 1995-11-17 Expression plasmid for yeast
EP95937777A EP0792368A1 (en) 1994-11-18 1995-11-17 Expression plasmids for yeasts

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19944441197 DE4441197C1 (en) 1994-11-18 1994-11-18 Schizosaccharomyces pombe expression plasmids
DEP4441197.9 1994-11-18

Publications (1)

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

Family

ID=6533638

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE1995/001604 Ceased WO1996016181A1 (en) 1994-11-18 1995-11-17 Expression plasmids for yeasts

Country Status (4)

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

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002491A1 (en) * 1995-07-04 1997-01-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Process for detecting specific antibodies acting against hpv proteins
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 (en) * 1985-10-25 1987-05-06 Zymogenetics, Inc. Method of using bar1 for secreting foreign proteins

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 (en) * 1985-10-25 1987-05-06 Zymogenetics, Inc. Method of using bar1 for secreting foreign proteins

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 (en) * 1995-07-04 1997-01-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Process for detecting specific antibodies acting against hpv proteins
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 (en) 1998-09-02
DE4441197C1 (en) 1996-03-28
EP0792368A1 (en) 1997-09-03

Similar Documents

Publication Publication Date Title
DE3546806C2 (en)
DE69233008T2 (en) Fusion of peptides and proteins with thioredoxin and thioredoxin-like molecules
DE69128285T2 (en) EXPRESSION OF RECOMBINANT POLYPEPTIDES WITH IMPROVED POSSIBILITY TO CLEAN
DE3485810T2 (en) METHOD FOR PRODUCING A RECOMBINANT BACULOVIRUS EXPRESSION VECTOR.
DE3789873T2 (en) IMMUNAFFINITY CLEANING SYSTEM.
DE69133012T2 (en) Method and composition for increasing the accumulation of squalene and specific sterols in yeast
DE3783191T2 (en) METHOD AND MEANS FOR PRODUCING A PROTEIN WITH THE SAME IGG SPECIFICITY AS PROTEIN G.
DD203331A5 (en) PROCESS FOR PRODUCING A POLYPEPTIDE
DE69636536T2 (en) Secretion signal gene and expression vector containing it
DE3752063T2 (en) Functional DNA block and plasmid coding for hirudin, transformed yeast by a process for the production of hirudin.
DE3587875T3 (en) RECOMBINANT METHOD FOR THE PRODUCTION OF SERINE PROTEASE INHIBITORS AND DNA SEQUENCES THEREFOR.
EP1136500A2 (en) Seroreactive regions from the HPV proteins E1 and E2
DE69013811T2 (en) Process for the production of antiviral protein using E. coli transformant, the gene coding for this protein and E. coli vector used in this process.
EP1664306A2 (en) Secretion of proteins from yeasts
DE60215315T2 (en) USE OF FUSION PROTEINS, THE N-TERMINAL PART OF WHICH IS A HIRUDIN DERIVATIVE, FOR THE PRODUCTION OF RECOMBINANT PROTEINS BY SECRETION BY HEALING
DE3641040C2 (en)
EP1216259B1 (en) Signal sequences for the production of leu-hirudine via secretion by e. coli in a culture medium
EP0100521B1 (en) Process for the production of a herpes antigen, appropriate means therefor and a process for its production, and the use of this antigen
EP1025253B1 (en) Positive-negative selection for homologous recombination
DE69506691T2 (en) Expression cassette containing a promoter derived from the Schizosaccharomyces pombe Pho4 gene
DE10108100A1 (en) Use of super-secretable peptides in processes for their preparation and parallel improvement of the export of one or more other polypeptides of interest
DE10196565B4 (en) Recombinant human parathyroid hormone producing yeast transformant and method for producing the hormone
WO1988000238A1 (en) Process for the genetic production of proteins by using of gram-negative host cells
DE3689918T2 (en) Procedure for using BAR1 for foreign protein secretion.
DE69933875T2 (en) PROTEIN ADMINISTRATION SYSTEM USING SIMILAR PARTICLES TO HUMAN PAPILLOMA VIRUS.

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1995937777

Country of ref document: EP

ENP Entry into the national phase

Ref country code: US

Ref document number: 1997 849267

Date of ref document: 19970828

Kind code of ref document: A

Format of ref document f/p: F

WWP Wipo information: published in national office

Ref document number: 1995937777

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1995937777

Country of ref document: EP