Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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Böldicke, T. et al.

Böldicke, Struck, Schaper, Tegge, Sobek, Villbrandt, Lankenau, Böcher,

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.[1]

References

A new peptide-affinity tag for the detection and affinity purification of recombinant proteins with a monoclonal antibody. Böldicke, T., Struck, F., Schaper, F., Tegge, W., Sobek, H., Villbrandt, B., Lankenau, P., Böcher, M. J. Immunol. Methods (2000) [Pubmed]

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