WO1999006577A2 - Agent for expressing and detecting a fusion polypeptide with an hpol-epitope and a polypeptide - Google Patents

Abstract

An expression vector is disclosed that codes for a fusion polypeptide with an HPOL-epitope and a polypeptide. Also disclosed is an antibody directed against such a fusion polypeptide, its use and a kit suitable for expressing and detecting the fusion polypeptide.

Description

Means for the expression and detection of a fusion polypeptide comprising an HPOL epitope and a polypeptide

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.

The expression of a polypeptide is still a challenge. This is particularly true if the expressed polypeptide is also to be detected. For this purpose, 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. Previous methods for the expression of a polypeptide and its detection often do not lead to satisfactory results.

The present invention is therefore based on the object of providing an agent with which a polypeptide can be expressed and, if appropriate, detected.

According to the invention this is achieved by the subject matter of the claims.

In particular, this is achieved by an expression vector coding for a fusion polypeptide comprising an HPOL epitope and a polypeptide, and by an antibody directed against the fusion polypeptide. The inventors have found that between amino acids 675 and 684 of the herpes simplex virus type I DNA polymerase there is an epitope (HPOL 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:

Glu-Glu-Gly-Gly-Gly-Glu-Arg-Glu-Pro-Glu

According to the invention, 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

A) For N-terminal tagging

BamHI Ncol Nrul

5 '-GGATCCACCATGGAGGAAC ^ TGGTGGTGAACGTC5Ai CCTGAGATCGAGGGTCGCGA- 3' 3 '-CCTAGGTGGTACCTCCTTCCACCACCACTTGCACTTGGACTCTAGCTCCCAGCGCT-5' fl

B) For internal and C-terminal tagging

For insertion in different reading frames, three versions:

EcoRI Nrul Bglll Xbal

5 '-GAATTCATCGAGGGTCGCGΆGGAAGGTGGTGGTGAACGTGAACCTGAGATCTAGA-3' 3 '-CTTAAGTAGCTCCCAGCGCTCCTTCCACCACCACTTGCACTTGGACTCTAGATCT-5'

EcoRI Nrul Bglll Xbal

5 '-GAATTCCATCGAGGGTCGCGAGGAAGGT.ΓGTGGTGAA.CGTGAACCTGAGATCTAGA-3' 3 '-CTTAAGGTAGCTCCCAGCGCTCCTTCC-ACCACCACTTGCACTTGGACTCTAGATCT-5'

EcoRI Nrul Bglll Xbal

5 '-GAATTCCCATCGAGGGTCGCGAGGAAGGTGGTGGTGAACGTGAACCTGAGATCTAGA-3' 3 '-CTTAAGGGTAGCTCCC1AGCGCTCCTTCCACCACCACTTGCACTTGGACTCTAGATCT-5'

ü Spa of factor Xa at the protein level

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. In the case of an expression vector for E. coli, these are e.g. B. pGEMEX, pUC derivatives, pGEX-2T, pET3b, pQE8 and pQE42, the latter being preferred. For expression in yeast e.g. to call pY100 and Ycpad l, while for expression in 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 person skilled in the art knows in what way 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.

Furthermore, 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. If 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. For the monoclonal antibodies, animal spleen cells are fused with myeloma cells.

Furthermore, 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.

For the production of synthetic antibodies e.g. from monoclonal antibodies obtained above. For this purpose it is advisable to analyze the antigen binding regions of the monoclonal antibodies and to identify the parts that are necessary and not necessary for the above recognition. The necessary parts can then be modified and the unnecessary parts can be completely or partially eliminated or replaced with parts that give the antibodies further favorable properties. Parts outside the binding regions of the antibodies can also be modified, eliminated or replaced. The person skilled in the art knows that DNA recombination technology is particularly suitable for the above measures. He is very familiar with this.

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. For this purpose, 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. 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. For this purpose, it is favorable to incubate the antibody-bound fusion polypeptides with biotinylated Xa factor protease and to clean the polypeptides obtained from the protease by incubation with streptavidin-Sepharose.

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.

Another object of the present invention is a kit. This kit 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.

Brief description of the drawing:

1 shows the construction of the vector pQE42PE, which expresses C-terminal HPOL-tagged dihydrofolate reductase (DHFR). For this, a

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.

The present invention is further illustrated by the following examples.

Example 1: Production of monoclonal antibodies

Mice were used for immunization. Purified HPOL fusion protein from vector pEX1051-transformed E. coli C600 cells of strain 537 was used as antigen (Knopf et al., Biochim. Biophys. Acta 951, pp. 298-314, 1,988). This was dissolved in PBS buffer.

Immunization and booster scheme:

Day 1: 50 / l (= 10 μg) HPOL fusion protein

50 μ \ PBS (phosphate-buffered saline) 200 μl Freund's adjuvant complete

300 ul mix

200 ul of the mix was injected into a mouse Day 30: 50 μl (= 10 μg) HPOL fusion protein

70 ul PBS 1 20 ul Freund's adjuvant incomplete

240 ul mix

200 ul of the mix was injected into the above mouse.

Day 60: 50 μl (= 10 μg) HPOL fusion protein

1 35 / l PBS 1 1 5 μl Freund's adjuvant incomplete

300 ul mix

200 ul of the mix was injected into the above mouse.

Day 90: 50 μl (= 10 μg) HPOL fusion protein

250 ul PBS

300 ul mix

200 ul of the mix was injected into the above mouse.

Day 1 80: 1 50 μl (= 20 μg) HPOL fusion protein 1 50 μl Freund's adjuvant complete

300 ul mix

200 ul of the mix was injected into the above mouse.

On day 200, the 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.

Example 2: Expression of an HPOL-tagged dihydrofolate reductase (DHFR)

After an intermediate cloning step in pUC1 8, 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. For expression analysis, pQE42 (= control vector) - transformed cells were also used and cell extracts were separated after 4 hours of growth in the presence and absence of 2mM IPTG using SDS-PAGE. After the Coomassi staining (Figure 2A, lane b, d), the recombinant DHFR proteins were overexpressed to the same extent. 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. To test the HPOL antibody / TAG detection, 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. The results of the immunostaining show that only protein bands in the gel lanes were stained with pQE42PE-transformed cell extracts, the 26.6 kDa band was mostly stained, even at an HPOL concentration of 5.5 ng / ml in gel lane c, ie a 26.6 kDa band was stained in the non-induced pQE42PE extract, which could not be represented by the Coomassie staining. This proves a high level of detection sensitivity and low cross-reaction for the HPOL-antibody / HPOL-antigen combination.

Example 3: Detection of HPOL fusion polypeptides by antibodies according to the invention by means of ELISA

100 μl each with 20 ng HPOL-DHFR fusion polypeptide according to Example 2 or lysate protein from E. coli, herpes virus type 2 protein, cytomegalovirus protein and Epstein-Barr protein were placed in a 96-hole plate per hole. Virus protein pipetted in as controls. After overnight incubation at 4 ° C, 3 short washing steps with PBS followed. The free binding sites of the polymeric carrier were then blocked by incubation for 1 hour with 2% skim milk powder in PBS at 37 ° C. An antibody diluted 1:10 or 1:50 according to the invention was incubated on the plate at 37 ° C. for 1 hour. After 8 washing steps with PBS, a peroxidase-coupled goat anti-mouse antibody (from Dianova) was added. After 30 minutes of incubation at 37 ° C., 8 washing steps followed and then the peroxidase detection reaction with developing solution (50 mM sodium acetate, 0.4 mM 3,3 ', 5,5'-tetramethylbenzidine dihydrochloride, 4.4 mM H) ₂ O ₂ ) at room temperature until color development was visible.

It was found that an antibody according to the invention specifically recognizes HPOL fusion polypeptides, but not a polypeptide without an HPOL component.

Example 4: Purification of HPOL-tagged proteins using an immunoaffinity resin

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.

To recover proteins that no longer contained HPOL-TAG under non-denaturing conditions, the 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.

Claims

K 2434Patent claims 1 . Expression vector coding for an HPOL epitope and a fusion polypeptide comprising a polypeptide, the HPOL epitope being the amino acid sequence Glu-Glu-Gly-Gly-Gly-Glu-Arg-Glu-Pro-Glu or a sequence different therefrom by one or more amino acids. 2. Expression vector according to claim 1, wherein the HPOL epitope is encoded by one of the following DNA sequences: A) For N-Teπninales Taggiπg Ba HI Ncol Nrul 5 '-GGATCC ^ CC ^ TGGAGGAA ^ TGGTGGTGI ^ CGTGAACCTGAGATCGAGGGTCGCGA- 3' 3 '-CCTAGGTGGTACCTCCTTCCACCACCACTTGCACTTGGACTCTAGCTCCCAGCGCT- 5' fϊ B) For internal and C-terminal Taggiπg For insertion in different reading frames, three versions: EcoRI Nrul Bglll Xbal 5 '-GÄATTCATCGAGGGTCGCGAGGAAGGTGGTGGTGAACGTGAACCTGAGATCTAGA-3' 3 '-CTTAAGTAGCTCCCAGCGCTCCTTCCACCACCACTTGCACTTGGACTCTAGATCT-5' EcoRI Nrul Bglll Xbal 5 '-G ATTCCATCGAGGGTCGCGÄGAAGG G GGTGAACGT ^ AACC GAG TCT GA-3 ^, 3' -CTTAAGGTAGCTCCCAGCGCTCCTTCCACCACCACTTGCACTTGGACTCTAGATCT-5 ' EcoRI Nrul Bglll Xbal 5 '-GAATTCCCATCGAGGGTCGCGAGGAAGGTGGTGGTGAACGTGAACCTGAGATCTAGA-3' 3 '-CTTi ^ GGGTAGCTCCCAGCGCTCCTTCCACCACCACTTGCACTTGGACTCTAGATCT-5' n 1Ϊ cleavage site of factor Xa at the protein level 3. Expression vector according to claim 1 or 2, wherein the HPOL epitope is present at the N- or C-terminus of the polypeptide. 4. Fusion polypeptide encoded by an expression vector according to one of claims 1 -3. 5. Antibody directed against a fusion polypeptide according to any one of claims 1-4. 6. The antibody of claim 5, wherein the antibody is directed against an HPOL epitope, wherein the HPOL epitope is the amino acid sequence Glu-Glu-Gly-Gly-Gly-Glu-Arg-Glu-Pro-Glu or a sequence different therefrom by one or more amino acids. 7. Antibody according to claim 5 or 6, wherein the antibody is monoclonal. 8. Use of the antibody according to any one of claims 5-7 in a detection method for a fusion polypeptide having an HPOL epitope. 9. Use according to claim 8, wherein the detection method is a Western blot, an ELISA, an immunofluorescence or an immunoprecipitation. 10. Use of the antibody according to any one of claims 5-7 for the purification of HPOL-tagged fusion polypeptides. 1 1. Use of the antibody according to any one of claims 5-7 for the diagnosis of HSV-1 infections. 2. Kit comprising an expression vector according to one of claims 1 -3, and / or an antibody according to any one of claims 5-7, and / or a DNA coding for the HPOL epitope, in particular one of Table 1, and common excipients such as buffers, carriers, marker compounds and Controls.