DE10019473A1 - Polypeptide tags for the detection and purification of polypeptides - Google Patents

Abstract

The present invention relates to polypeptide tags that can be used for the isolation, purification and detection of polypeptides. The present invention relates in particular to expression vectors which encode a fusion polypeptide which comprises a tag54 epitope and a desired polypeptide, and to the use of such expression vectors for expressing the fusion polypeptides. Furthermore, the invention relates to antibodies against the fusion peptides generated with the described polypeptide tag, their use for the isolation, purification and detection of the fusion peptides, and the kits suitable for this purpose.

Description

The present invention relates to polypeptide tags used for isolation Purification and detection of polypeptides can be used. The present invention also relates to expression vectors that are a fusion encode a polypeptide comprising a tag54 epitope and a desired polypeptide, and the use of such expression vectors for the expression of the fusion polypeptides.

The expression of polypeptides remains a challenge. This is especially true when the expressed polypeptide is used regardless of the th expression system is to be detected and purified.

For the analysis or purification of polypeptides, the technique of so-called "epitope taggings" used, in which by means of molecular biological Techniques attached a particular epitope to a polypeptide of interest and that fusion peptide formed during expression then against the "tag" directed antibody is isolated. The primary sequence of any Polypeptide using recombinant techniques around a few amino acids which are specifically recognized by the antibody. Decisive for Success is therefore the presence of a specific and high affinity Antibody with a known recognition sequence, the sequence without cross-reactiv specifically with other protein sequences.

To date, a residue of six histidine residues (His6) has been used as such a "tag" set. However, this "tag" has the disadvantage that none against it are high affine antibodies can be produced and that the existing antibodies with different protein material are cross-reactive.  

It is therefore an object of the present invention to provide an agent to provide, which avoids the disadvantages of the known "tags" and with the Polypeptides can be expressed and specifically detected.

This problem is solved by a polypeptide tag which has the following amino acid sequence:
-Lys-Asp-Trp-Glu-His-Leu- (SEQ ID No. 2)
or an amino acid sequence derived from the sequence given above, in which the three-dimensional or tertiary structure is essentially retained. The sequence derived from the specified amino acid sequence can be obtained by substitution and / or inversions of one or more amino acids. According to a preferred embodiment, the polypeptide tag has the following amino acid sequence: Lys-His-Ile-Lys-Asp-Trp-Glu-His-Leu-Glu-Glu-Phe (SEQ ID No. 4) or one of the above indicated sequence derived amino acid sequence in which the three-dimensional or tertiary structure is essentially preserved.

In the figures,

Fig. 1 shows schematically a vector (pSSH1) for the expression of fusion peptides in plants; and

Fig. 2 shows schematically an expression vector (pET22b) for fusion peptides in bacteria.

The inventors named here have now found that a certain area of the TMV 54K protein (cf. Padgett et al. "Analysis of a Tobacco Mosaic Virus Strain capable of overcoming N gene-mediated resistance "; Plant Cell 5 (1993), 577- 586), in particular the amino acid sequence between amino acids 126 and 139 of the TMV 54K protein is ideal as a "day" because it is against this area  high affinity antibodies can be produced and cross-reactive with cellular epitopes of bacterial and eukaryotic origin essentially is not present.

In order to use the amino acid sequences listed above as a "tag" for a polypeptide of interest, a nucleotide sequence encoding the "tag" is inserted into an expression vector which preferably has the following base sequence:
AAG GAC TGG GAG CAC (SEQ ID No. 4)
respectively.
AAG CAC ATC AAG GAC TGG GAG CAC CTC GAA GAG TTC (SEQ ID No. 5)
or a sequence derived from the specified sequence which encodes a "tag" according to the invention.

A expression encoding a polypeptide of interest is also inserted into the expression vector DNA sequence inserted such that both DNA sequences are uniform Form reading frame and a fusion polypeptide is produced that the polypeptide of interest and that contains "tag". The "tag" can be at the N- or C-terminal End, or also within the fusion polypeptide and possibly from the Polypeptide through a spacer consisting of one or more amino acids be spaced.

The expression vectors usually contain the elements that are usual for them, such as Promoter, polylinker to facilitate the cloning of foreign sequences, Leader sequences to control the fusion peptide out of the cell, etc. The Vectors are considered taking into account the requirements of the desired Expression system selected. For expression in E. coli, for example refer to the vectors pGEMEX, pUC derivatives, pGEX-2T, pET derivatives, pQE8 are used, all of which are already widely used as expression vectors come.  

For yeasts, for example, the vectors are pY100, Ycpad1, pPIC9K and pPICz can be used, while for expression in animal cells the vectors pKCR, pEFBOS, cDM8 and pCEV4 can be used. For an expression in The vector pAL76 and pSS derivatives can be planted (see for example Kay R. et al., Science 236 (1987), 1299-1302) can be used during expression in insect cells using, for example, the baculovirus expression vector pAcSGNT-A can be achieved.

The person skilled in the art can, based on his general specialist knowledge, the respective DNA sequences for the polypeptide tag or the polypeptide of interest in the Insert expression vector so that expression of the fusion peptide is achieved or can be optimized. It may be preferred that the polypeptide DNA coding sequence before inserting the DNA for the polypeptide into the expression vector is inserted. It is also clear to the person skilled in the art that due to the degeneration of the genetic code changes the DNA in such a way can, for example, a codon-optimized DNA sequence for the respective To provide expression system.

Methods and cells are known to the person skilled in the art to produce fusion polypeptides to express. Examples of such cells include the E. coli strains HB101, DH1, JM101, 1M109 and BL21, the yeast Saccharomyces cerevisae and Pichia pastoris, the animal cell lines L 3T3, FM3A, CHO, COS, Vero and HeLa, and the insect cell line Sf9. Furthermore, Monocotyledoneae and Dicotyledoneae, can be used both as an intact plant and in cell suspension culture.

Furthermore, the present invention provides one against the invention Polypeptide-directed antibodies.

With the help of antibodies that are specific to the extended protein content are directed, for example, the molecular weight of a protein can be determined  its cellular location, post-translational modifications or Interaction with other factors without the need for protein-specific antibodies must be present, as well as a purification of proteins easily performed become (affinity chromatography).

The antibody can be a polyclonal or monoclonal antibody or the "Tag" binding fragment thereof, being a monoclonal antibody is preferred. This can be done by customary methods known to those skilled in the art getting produced. Should polyclonal or monoclonal antibodies be produced then it is advantageous to preserve animals, especially rabbits polyclonal antibodies, as well as mice for obtaining monoclonal antibodies with the corresponding fusion polypeptide or a fragment thereof that the contains corresponding epitope, or to immunize with the purified epitope. The animals can be boosted further using the same antigens. In addition other fusion peptides containing the inventive polypeptide tag or a mixture of the fusion polypeptide used first and one or several new fusion polypeptides can be used to boost. The so Polyclonal antibodies obtained can then be obtained from the animal serum be won.

Spleen cells from the immunized animals are used to obtain monoclonal antibodies fused to myeloma cells according to known methods.

In addition, an antibody according to the invention can be synthetic, where appropriate Parts that are not necessary for the recognition of the polypeptide tag according to the invention are necessary, missing in whole or in part, or replacing these parts with others are which give the antibody further favorable properties.

For example, synthetic antibodies can be prepared as described above obtained monoclonal antibodies. For this, the Antigen binding regions of the monoclonal antibodies were analyzed and those for  Detection of necessary and unnecessary parts can be identified. The need Agile parts can then be modified and all or part of the unnecessary parts wisely eliminated or replaced with parts that further the antibodies give favorable properties. Parts can also be outside the binding regions of the antibodies are modified, eliminated or replaced.

The antibodies according to the invention are distinguished by the fact that they use the Detect and bind any fusion polypeptides according to the invention can. The antibodies are therefore suitable for the rapid detection of expression such fusion polypeptides, which are carried out in any method can, especially in a Western blot, an ELISA, an immunoprecipitation or immunofluorescence. For this purpose, the antibodies according to the invention be marked or in combination with marked, directed against them Antibodies are used.

The antibodies according to the invention can be used for this to purify suitable fusion polypeptides from, for example, cell extracts. A bin the antibodies can be added to the "tag" in a wide pH range, for example in a range from about pH 3 to about 10.

Furthermore, the present invention provides a kit that contains an expression vector contains with which a desired polypeptide in a particular system can be expressed. The expression vector preferably already contains the DNA sequence for the polypeptide tag according to the invention, so that the person skilled in the art only has to introduce the polypeptide of interest into the expression vector. Alternatively, the DNA sequence encoding the polypeptide tag can be separately provided be seen so that the user can decide at which point of the resulting fusion polypeptide the "tag" should appear. Furthermore, the Kit an antibody directed against the tag according to the invention as well as usual Excipients such as buffers, carriers, marker compounds and controls.  

The invention will now be further explained with reference to the examples. Where Techniques and conditions were not specifically explained, as in Sambrook, J., et al. (1989). Molecular cloning: A laboratory manual, 2nd Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

example 1 Production of recombinant antibodies

Using hybridoma technology (Köhler G. and Milstein C., Nature 256 (1975), 52-53 Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity) a monoclonal antibody, mAb 54-1, was raised against the TMV 54K viral protein generated. Female Balb / c mice were used for the immunization, whereby as Antigen is a fusion protein of TMV 54K with GST (glutathione-S-transferase) was used.

After isolating the mRNA from antibody-secreting hybridoma cell lines according to conventional techniques, the variable domains of the heavy chain (V _H ) and the light chain (V _L ) were cloned by RT-PCR using the following oligonucleotides:

V _H amplification 5 'primer

5'-CAT GCC ATG ACT CGC GGC CCA GCC GGC CAT GGC CCA GGT TAC TCT RAA AGW GTS TGG CC-3 '(SEQ ID No. 6)

3 'primer

5'-TGM RGA GAC GGT GAC CAG RGT C-3 '(SEQ ID No. 7)

V _L amplification 5 'primer

5'-CTA GTG GTA CTC CAC GCG GCC GCG TCG ACA GCM CGT TTC AGY TCC ARY TT-3 '(SEQ ID No. 8)

3 'primer

5'-CAT GCC ATG ACT CGC GGC GCG CCT GAC ATT GTG MTG WCH CAG TCT CCA-3 '(SEQ ID No. 9)
according to the IUPAC Convention from 1984:
G: guanine; A: adenine; T: thymine; C: cytosine
R: adenine or guanine
Y: thymine or cytosine
W: adenine or thymine
S: guanine or cytosine
M: adenine or cytosine
K: Guanine or Thymine
H: adenine or thymine or cytosine
B: guanine or cytosine or thymine
V: guanine or adenine or cytosine
D: guanine or adenine or thymine
N: guanine or adenine or thymine or cytosine

The nucleotide sequences thus obtained were in the form of so-called "single chain antibodies "(scFv) expressed in E. coli (Winter G. and Milstein C., Nature 349 (1991), 293-299).

Then both the sequence of the monoclonal antibody and of the scFv antibody elucidated (scFv54-1, SEQ ID NO. 1), or via checks, whereby no specificity difference could be shown.

Example 2 Identification of the recognized epitope

According to the method of Cortese et al. ("Identification of biologically active peptides using random libraries displayed on phage ", Curr. Opin. Biotechnol. 6 (1995), 73-80), a phage library was created to use the so-called Peptide displays identify linear as well as structural epitopes. The created Phage libraries express an undetermined 9 amino acid peptide  at the N-terminus of Lambda gene VIII. A library presented linear peptides, the other closed peptides.

To identify peptides recognized by the monoclonal antibody mAb 54-1 obtained in Example 1, the libraries were hybridized with the affinity-purified monoclonal antibody (20 µg per round of selection) using a standard protocol (Cortese et al., Supra) . In the first round of the selection, 5 × 10 ¹² phages in 1 ml PBS were incubated with immobilized antigen (16 hours, 4 ° C.). After washing (15 × 4 ml PBST and 5 × 4 ml PBS), binding phages were eluted with 1 ml Glycine-HCl pH 2.2 and 0.1% (w / v) BSA (10 min, 20 ° C) Neutralized 60 µl 2M Tris and then used to infect E. coli. The phage titer was then determined by plating 100 .mu.l infected cells on 2x TY-amp plates. Enrichment factors were determined using a parallel BSA control. Monoclonal phages from the third round of selection were checked for their reactivity in the phage ELISA.

The positively selected phages obtained in this way were sequenced. Each received The sequences were compared and the consensus sequence was determined.

The results are summarized in Table 1:

Table 1

When comparing the consensus sequence according to Table 1 with the sequence of the TMV polypeptide 54K was able to find the corresponding site recognized by mAK 54-1 derived in the TMV 54K polypeptide.  

Example 3

Incorporation of the epitope into recombinant proteins and detection

According to the determination of mAk 54-1 in TMV 54K described in Example 2 recognized epitopes, the DNA sequences were abge according to the genetic code directs and corresponding oligonucleotides in a classic polymerase chain reaction used to amplify the tag54 epitope.

These epitopes have been used as a means of detection for plants and Bacterial-derived recombinant proteins built into commercially available vectors.

a. Expression in plants

The epitope tag-54 was fused "in frame" to the C-terminus of the antibody fragment scFv24. For this purpose, the C-terminal c-myc and His6 fragment was removed from the vector pscFv24CM (Zimmermann et. Al. Molecular Breeding 4 (1998), 369-379) and replaced by the SalI / XbaI tag-54 fragment ( FIG. 3 ). The complete expression cassette in the plant expression vector pSSH1 (Kay R. et al., Science 236 (1987), 1299-1302) contained the CaMV 355 promoter with the double enhancer, the 5 'untranslated region of the chalcone synthase, the signal peptide sequence of the light chain of the monoclonal antibody 24 (Voss et al., Molecular Breeding 1 (1995), 39-50), the scFv24 (Zimmermann et. al. supra), the tag-54, which has a glycine-glycine-glycine-glycine-serine Linker in reading frame with the scFv24 was fused, and the 3 'untranslated region and termination sequence of CaMV. This construct, pscFv24-S4tag, allowed transport and accumulation of the recombinant protein in apoplasts of the plant cell.

A second plant expression vector, pscFv24-54tagK, located at the C-terminus of the coding region additionally contained the amino acid sequence KDEL the accumulation of the recombinant protein in the endoplasmic reticulum Plant cell.  

Both constructs were transient in Nicotiana tabaccum cv. Petite Havana SR1 Leaf transformed and after three days of incubation, the soluble proteins by grinding the tobacco leaves in mortars in the extraction buffer (200 mM Tris HCl pH 7.5; 5mM EDTA, Tween 20; 0.1 mM DTT) isolated.

The expression and enrichment of the recombinant proteins was determined by means of ELISA and Western blot on Tag-54 using the antibody mAb 54-1 proven. In the ELISA, 1,375 ng for the construct pscFv24-54tag and for the construct pscFv24-54tagK 26,480 ng functional scFv24 per gram Sheet material can be recorded.

The following components were analyzed in the ELISA in the order given uses: 1) polyclonal chicken anti TMV antibodies (41 mg / ml diluted 1: 5000), 2) 1 µg / ml TMV vulgare, 3) soluble from isolated tobacco leaves Protein, 4) rabbit polyclonal antibodies specific to the monoclonal Bind antibody 24 (diluted 1: 5000), 5) labeled with alkaline phosphatase Goat anti rabbit antibody (0.6 mg / ml diluted 1: 5000), 6) phosphatase substrate p-nitrophenyl phosphate.

Both recombinant proteins, scFv24-54tag and scFv24- 54tagK, can be specifically detected. For this, 20 µl of the tobacco leaves were used isolated soluble proteins separated on an SDS polyacrylamide gel Nitrocellulose membrane transferred (Sambrook, supra) and the recombinant Proteins scFv24-54tag and scFv24-54tagK detected as follows: 1) mAb 54-1 (0.8 mg / ml and diluted 1: 5000), 2) an alkaline phosphatase labeled Goat anti mouse antibody (0.6 mg / ml diluted 1: 5000), 3) staining reagent NBT / BCIP.  

b. Expression in bacteria

The vector pET22b (Novagen, Ca, USA) was modified in such a way that it enables "in-frame" cloning of the GST protein in fusion with the tag-54 ( FIG. 4). First, the tag54 epitope was cloned into the bacterial expression vector pGEX5x-3 (Pharmacia, SE) and expressed as a GST (glutathione-S-transferase) fusion peptide (GST-tag54) using transformed E. coli BL21 cells. For this purpose, the E. coli cells were grown at 30 ° C. to an OD _{600 nm} of 0.8 with shaking and then induced with 1 mM IPTG. After 4 hours, the cells were collected by centrifugation (4000 × g, 4 ° C). The cells were lysed using ultrasound, whereupon 30 μg of protein A purified monoclonal antibody mAK 54-1 were added to 10 ml of bacterial lysate.

After incubation for 20 min, the GST-Tag54 / mAK 54-1 formed Association product isolated using a GST affinity purification (Pharmacia, SE) and eluted from the matrix using reduced glutathione. The complex was separated by SDS-PAA gel electrophoresis gel electrophoresis, where on the Coomasie blue stained gel both the light and heavy antibodies Chain as well as the GST-tag54 protein were detected.  

SEQUENCE LOG

Claims (15)

1. Polypeptide tag that has the following amino acid sequence
Lys-Asp-Trp-Glu-His-Leu
or a sequence derived from the above sequence by replacing one or more amino acids, in which the three-dimensional or tertiary structure is essentially retained. 2. The polypeptide tag according to claim 1, which has the following amino acid sequence
Lys-His-Ile-Lys-Asp-Trp-Glu-His-Leu-Glu-Glu-Phe
or a sequence derived from the above sequence by replacing one or more amino acids, in which the three-dimensional or tertiary structure is essentially retained. 3. Expression vector encoding a fusion polypeptide that is a desired one A polypeptide and a polypeptide tag according to any one of claims 1 or 2. 4. Expression vector according to claim 3, wherein the polypeptide tag of the ge desired polypeptide is separated by a spacer. 5. Expression vector according to claim 3 or 4, wherein the polypeptide tag is encoded by the following DNA sequence:
AAG GAC TGG GAG CAC
or a different DNA sequence encoding a polypeptide tag according to claim 1. 6. Expression vector according to claim 3 or 4, wherein the polypeptide tag is encoded by the following DNA sequence:
AAG CAC ATC AAG GAC TGG GAG CAC CTC GAA GAG TTC
or a different DNA sequence encoding a polypeptide tag according to claim 2. 7. Expression vector according to one of claims 3 to 6, wherein the polypeptide tag is present at the N or C terminus or within the polypeptide. 8. Fusion polypeptide derived from an expression vector according to any one of the claims 3 to 7 is encoded. 9. Antibody or a fragment thereof that is against a polypeptide tag after a of claims 1 or 2 is directed. 10. The antibody of claim 9, wherein the antibody is monoclonal. 11. Use of a polypeptide tag according to claim 1 or 2 for isolation and / or for the detection of a polypeptide. 12. Use of an expression vector according to one of claims 3 to 7 for Preparation and / or isolation of a polypeptide. 13. Use of an antibody according to claim 9 or 10 for cleaning and / or Isolation and / or detection of a polypeptide. 14. Use of an antibody according to one of claims 9 or 10 for Diagnosis of TMV infections.   15. A kit comprising an expression vector according to any one of claims 3 to 7 and optionally an antibody according to one of claims 9 or 10.