A new peptide-affinity tag for the detection and affinity purification of recombinant proteins with a monoclonal antibody.
A monoclonal anti-peptide antibody (2E11) was raised against the synthetic peptide 38 (C-L-D-K-S-G-L-P-S-D-R-F-F-A) representing a part of the variable region of the Vbeta 6.2 T-cell receptor. This mAb (IgG(1), kappa light chain) bound very specifically to peptide 38 as shown by ELISA but did not recognize the corresponding native Vbeta 6.2 T-cell receptor on T-cells. For epitope analysis, overlapping peptides of 4-10 amino acids in length corresponding to the sequence of peptide 38 were synthesized and assayed by SPOT synthesis on cellulose sheets. The shortest peptide recognized was L-P-S-D-R. The specificity of mAb 2E11 was examined with 100 different peptides comprising other parts of the different variable Vbeta domains of the human T-cell receptor that do not include the epitope region L-P-S-D-R. None of these peptides were recognized. The chemical synthesis of a peptide with the sequence L-P-S-D-R on Sepharose beads allowed to efficiently purify the mAb 2E11 in a single step by affinity chromatography. An equilibrium binding constant of 4.9x10(6) l/mol was determined for mAb 2E11 by using rhodamine-green-labelled peptide 38 in fluorescence correlation spectroscopy. In order to demonstrate that peptide 38 can be used as an affinity-tag, it was fused to the carboxyl-terminus of interferon regulatory factor-1 (IRF-1). It could be shown that in vitro translated peptide 38 tagged IRF-1 was immunoprecipitated by the mAb 2E11 and that the fusion protein could be purified by immunoaffinity chromatography. Additionally peptide 38 was fused to the amino-terminus of the Taq polymerase. This recombinant protein was expressed in E. coli and specifically detected in a Dot blot and Western blot using mAb 2E11.
Böldicke
T
T
Gesellschaft für Biotechnologische Forschung mbH, Department of Applied Genetics, Braunschweig, Germany. tbo@gbf.de
Struck
F
F
Schaper
F
F
Tegge
W
W
Sobek
H
H
Villbrandt
B
B
Lankenau
P
P
Böcher
M
M
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D000939:Epitopes; D006801:Humans; D050856:Interferon Regulatory Factor-1; D009842:Oligopeptides / Q000235:genetics / Q000276:immunology*; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D009842:Oligopeptides; D010446:Peptide Fragments; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins