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WO1999006577A2 - Agent permettant d'exprimer et de mettre en evidence la presence d'un polypeptide de fusion comprenant un epitope hpol et un polypeptide - Google Patents

Agent permettant d'exprimer et de mettre en evidence la presence d'un polypeptide de fusion comprenant un epitope hpol et un polypeptide Download PDF

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Publication number
WO1999006577A2
WO1999006577A2 PCT/DE1998/002221 DE9802221W WO9906577A2 WO 1999006577 A2 WO1999006577 A2 WO 1999006577A2 DE 9802221 W DE9802221 W DE 9802221W WO 9906577 A2 WO9906577 A2 WO 9906577A2
Authority
WO
WIPO (PCT)
Prior art keywords
hpol
epitope
glu
antibody
polypeptide
Prior art date
Application number
PCT/DE1998/002221
Other languages
German (de)
English (en)
Other versions
WO1999006577A3 (fr
Inventor
Karl-Werner Knopf
Uwe Schreiner
Reiner Strick
Jutta Hansen
Original Assignee
Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Bayer Aktiengesellschaft
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 Stiftung des öffentlichen Rechts, Bayer Aktiengesellschaft filed Critical Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority to EP98948711A priority Critical patent/EP1002112A1/fr
Priority to JP2000505317A priority patent/JP2001512026A/ja
Publication of WO1999006577A2 publication Critical patent/WO1999006577A2/fr
Publication of WO1999006577A3 publication Critical patent/WO1999006577A3/fr

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    • 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)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to an expression vector which codes for a fusion polypeptide comprising an HPOL epitope and a polypeptide.
  • the invention further relates to an antibody directed against such a fusion polypeptide, its use and a kit suitable for expression and detection of the fusion polypeptide.
  • a polypeptide is still a challenge. This is particularly true if the expressed polypeptide is also to be detected.
  • methods are known in which a polypeptide is expressed in the form of a fusion polypeptide and this is detected by an antibody directed against the portion fused to the polypeptide.
  • Such a process includes e.g. the expression of a polypeptide in the form of a histidine fusion polypeptide with a portion of approx. 6 histidines at the C- or N-terminal end (His-TAG) of the polypeptide and the detection of the fusion polypeptide by an antibody directed against the histidine portion.
  • His-TAG C- or N-terminal end
  • the present invention is therefore based on the object of providing an agent with which a polypeptide can be expressed and, if appropriate, detected.
  • an expression vector coding for a fusion polypeptide comprising an HPOL epitope and a polypeptide, and by an antibody directed against the fusion polypeptide.
  • HPOL epitope an epitope recognizable by an antibody. It was also recognized that this epitope is ideally suited as a TAG for a polypeptide.
  • the HPOL epitope contains a high proportion of acidic amino acids and is therefore an ideal surface antigen that is easily accessible to an antibody directed against it.
  • the HPOL epitope found by the inventors comprises amino acids 675-684 of the HSV-1 DNA polymerase from the Angelotti strain (see Knopf et al., Nucleic Acids Res. 14 (1 986), pp. 8225-8226). These amino acids have the following sequence:
  • this amino acid sequence can also be changed by one or more amino acids, the functionality of the epitope being retained.
  • the changes in the amino acid sequence can be additions, deletions, substitutions and / or inversions of one or more amino acids.
  • the HPOL epitope and its changes are also the subject of the present invention.
  • the HPOL epitope can be used as a TAG for a polypeptide. For this purpose, it makes sense to insert a DNA coding for the epitope into an expression vector into which a DNA coding for the polypeptide is also inserted. Both DNAs are inserted in such a way that they are expressed as a fusion polypeptide. In such a case, the HPOL epitope can be present at the N- or C-terminal end or within the polypeptide. To express the fusion polypeptide, the DNA coding for the HPOL epitope preferably has the sequences given in Table 1: Table 1
  • Examples of the expression vector into which the DNAs coding for the HPOL epitope or the polypeptide can be inserted are known to the person skilled in the art.
  • these are e.g. B. pGEMEX, pUC derivatives, pGEX-2T, pET3b, pQE8 and pQE42, the latter being preferred.
  • yeast e.g. to call pY100 and Ycpad l
  • animal cells e.g. pKCR, pEFBOS, cDM8 and pCEV4 must be specified.
  • the baculovirus expression vector pAcSGNT-A is particularly suitable for expression in insect cells.
  • the DNAs for the HPOL epitope or the Polypeptide must be inserted into the expression vector in order to achieve the expression of the fusion polypeptide with the HPOL epitope at the C or N terminus or within the polypeptide. It is preferred for a C- or N-terminal HPOL epitope to insert the DNA coding for this into the expression vector before the DNA is inserted for the polypeptide. For an internal HPOL epitope, it is preferred to insert its coding DNA into the DNA for the polypeptide via suitable restriction sites and to insert the resulting construct into the expression vector.
  • the DNA encoding the polypeptide is not restricted. It can be of any type and length and can code for any polypeptide in natural form except for an HSV-1 DNA polymerase.
  • the person skilled in the art knows methods and cells in order to express the expression vector and the fusion polypeptide encoded by it.
  • Examples of such cells include the E. coli strains HB101, DH 1, x1 776, JM 101, JM 109, BL21 and SG 1 3009, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa and the insect cells Sf9.
  • An expression vector which codes for a fusion polypeptide comprising an HPOL epitope and a polypeptide, and the fusion polypeptide itself are also the subject of the present invention.
  • Another object of the present invention is an antibody directed against an above fusion polypeptide.
  • the antibody is preferably directed against the HPOL epitope.
  • the antibody can be a polyclonal or monoclonal antibody, with a monoclonal antibody being preferred.
  • the antibody can be obtained from any animal, with rabbits being preferred for a polyclonal antibody and rabbits being preferred for a monoclonal mouse.
  • a particularly preferred monoclonal antibody was submitted to the DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig) as 1051 c under DSM ACC2323 on July 29, 997 for patent filing in accordance with the Budapest Treaty.
  • the above antibodies can be produced by conventional methods.
  • polyclonal or monoclonal antibodies are to be produced, it is advantageous to immunize animals, in particular rabbits for the former and mice for the latter antibodies, with an HPOL fusion polypeptide or a fragment which contains the HPOL epitope, or the purified HPOL epitope .
  • the animals can be further boosted with the same or the same antigens.
  • Other HPOL fusion polypeptides or a combination of these and the preceding HPOL fusion polypeptide (s) can also be used for the booster.
  • the polyclonal antibodies can then be obtained from the serum of the animals.
  • animal spleen cells are fused with myeloma cells.
  • an antibody according to the invention can be synthetic, parts or parts which are not necessary for the above recognition being missing in whole or in part, or these parts being replaced by others which give the antibody further favorable properties.
  • Antibodies according to the invention are distinguished in that they recognize any fusion polypeptides which have an HPOL epitope.
  • the antibodies are therefore suitable for the rapid detection of the expression of such fusion polypeptides. This can be done in any detection method, especially in one Western blot, an ELISA, immunoprecipitation or immunofluorescence.
  • the antibodies according to the invention can, if appropriate, be labeled or be used in combination with labeled antibodies directed against them.
  • the antibodies are also suitable for purifying the above fusion polypeptides, for example from cell extracts. This can be done, for example, by coupling the antibodies to column material and binding the fusion polypeptides present in the cell extracts to the antibodies.
  • Such binding can take place in a wide pH range, in particular from pH 3-10.6.
  • Buffers with a 4M salt concentration can preferably be used to purify the antibody-bound fusion polypeptides.
  • the fusion polypeptides are eluted, for example, with 0.1 M glycine at pH 2.3-2.7.
  • the fusion polypeptides When using the sequences given in Table 1 for the HPOL epitope, the fusion polypeptides have an Xa factor cleavage site and can therefore be easily isolated without the HPOL epitope after elution.
  • Antibodies according to the invention are herpes type 1-specific and can therefore also be used for the specific diagnosis of an HSV-1 infection. They can be used for all immunoblot procedures (colony hybridization, dot blot, western blot etc.) as well as for immunoprecipitation, ELISA, immunohistochemistry and cytochemistry. Cross-reactions with proteins of prokaryotic and eukaryotic origin are not observed.
  • kits comprises: an expression vector which codes for a fusion polypeptide comprising an HPOL epitope and a polypeptide and / or an antibody which is directed against such a fusion polypeptide and / or a DNA which codes for the HPOL epitope, in particular one of table 1, and common excipients such as buffers, carriers, marker compounds and controls.
  • HPOL DNA cassette inserted into the BamHI and Smal site of pUC1 8 (a).
  • the HPOL-DNA cassette was then cut out using the restriction enzymes BamHI / Ecl l 3611 and inserted into the vector pQE42 opened with Bgl II and Smal.
  • the vector pQE42PE was obtained.
  • mice were sacrificed. Spleen cells were removed from them and fused with myeloma cells. Three monoclonal antibodies were obtained Characterization of these antibodies showed that in particular the antibody 1051 c, specified above, specifically recognizes the above HPOL epitope.
  • an HPOL-DNA cassette was inserted into the expression vector pQE42 (from Qiagen, Hilden), which contains the genetic information for dihydrofolate reductase (cf. FIG. 1).
  • the resulting vector pQE42PE was used to transform M1 5 [pREP4] cells.
  • the 24.8 kDA His-DHFR protein was enriched from the induced control vector-transformed cells and the 26.6 kDa HPOL-tagged His-DHFR from the induced pQE42PE-transformed cells.
  • an immunoblot was made from an SDS polyacrylamide gel carried out under identical conditions, the blot was cut into three parts and each blot part was immunostained with different antibody concentrations using standard methods.
  • Example 3 Detection of HPOL fusion polypeptides by antibodies according to the invention by means of ELISA
  • an antibody according to the invention specifically recognizes HPOL fusion polypeptides, but not a polypeptide without an HPOL component.
  • An antibody according to the invention was covalently bound to a resin and incubated with the cellular lysate, which contained the recombinant HPOL-tagged protein from Example 2, under customary conditions of immunoprecipitation and filled into a chromatography column. After immobilization, the resin was washed with a buffer containing up to 4M potassium or sodium chloride. This was followed by washing with a buffer of lower sanz concentration (up to 1 M). Partially native HPOL-tagged proteins could be eluted with 0.1 M glycine / HCl, pH 2.3-2.7. This buffer was also used for the reconstitution of the antibody column.
  • HPOL-tagged proteins were applied to the HPOL antibody column as before.
  • the washing steps were carried out as above and the immobilized proteins were incubated with biotinylated factor Xa proteinase.
  • the cleaved proteins of the eluate or supernatant were removed from the proteinase by streptavidin resin purification.

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Abstract

L'invention concerne un vecteur d'expression codant un polypeptide de fusion comprenant un épitope HPOL et un polypeptide. L'invention concerne en outre un anticorps dirigé à l'encontre d'un polypeptide de fusion de ce type, son utilisation, ainsi qu'un kit approprié à l'expression et à la mise en évidence du polypeptide de fusion.
PCT/DE1998/002221 1997-08-01 1998-07-29 Agent permettant d'exprimer et de mettre en evidence la presence d'un polypeptide de fusion comprenant un epitope hpol et un polypeptide WO1999006577A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP98948711A EP1002112A1 (fr) 1997-08-01 1998-07-29 Agent permettant d'exprimer et de mettre en evidence la presence d'un polypeptide de fusion comprenant un epitope hpol et un polypeptide
JP2000505317A JP2001512026A (ja) 1997-08-01 1998-07-29 Ｈｐｏｌエピトープとポリペプチドとの融合ポリペプチドの発現及び検出用薬剤

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19733389A DE19733389C2 (de) 1997-08-01 1997-08-01 Mittel zur Expression und zum Nachweis eines ein HPOL-Epitop und ein Polypeptid enthaltenden Fusionspolypeptids
DE19733389.3 1997-08-01

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WO1999006577A2 true WO1999006577A2 (fr) 1999-02-11
WO1999006577A3 WO1999006577A3 (fr) 1999-04-22

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PCT/DE1998/002221 WO1999006577A2 (fr) 1997-08-01 1998-07-29 Agent permettant d'exprimer et de mettre en evidence la presence d'un polypeptide de fusion comprenant un epitope hpol et un polypeptide

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EP (1) EP1002112A1 (fr)
JP (1) JP2001512026A (fr)
DE (1) DE19733389C2 (fr)
IT (1) ITMI981685A1 (fr)
WO (1) WO1999006577A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7105644B2 (en) 1997-07-08 2006-09-12 Human Genome Sciences, Inc. Secreted protein HHTLF25 antibodies

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5120639A (en) * 1989-04-03 1992-06-09 E. R. Squibb & Sons, Inc. Selective inhibition of dna polymerase
AUPN480095A0 (en) * 1995-08-15 1995-09-07 Commonwealth Scientific And Industrial Research Organisation Epitope tagging system

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7105644B2 (en) 1997-07-08 2006-09-12 Human Genome Sciences, Inc. Secreted protein HHTLF25 antibodies

Also Published As

Publication number Publication date
JP2001512026A (ja) 2001-08-21
DE19733389A1 (de) 1999-02-11
DE19733389C2 (de) 1999-08-19
EP1002112A1 (fr) 2000-05-24
ITMI981685A1 (it) 2000-01-21
ITMI981685A0 (it) 1998-07-21
WO1999006577A3 (fr) 1999-04-22

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