Detection of pET-vector encoded, recombinant S-tagged proteins using the monoclonal antibody ATOM-2

The 15-meric S-tag is a truncated form of the S-peptide, which builds toget her with the 103 amino acid large S-protein the whole ribonuclease S-protei n, Its small size and excessive solubility have made the S-tag an excellent fusion partner in the production of recombinant proteins, and a large vari ety of applications have been reported using the S-tag as a carrier. While S-tagged proteins were mostly detected and analyzed so far by use of their affinity to S-proteins, monoclonal antibodies (MAbs) for this tag have been not available. The generation of antibodies specific for S-tagged proteins is expected to broaden the range of applications of such S-tag fused recom binant proteins, and in this contest, a novel MAb termed ATOM-2 was generat ed that specifically binds S-tagged proteins, which have been expressed usi ng pET-vectors, Antigen specificity of ATOM-2 was confirmed in Western blot and enzyme-linked immunoadsorbent assay analysis, and using a series of am ino acid deletion mutants, the binding epitope of ATOM-2 was precisely mapp ed. This showed that ATOM-2 recognizes the C-terminal part of the 15-meric S-tag in context with a few residues of vector encoded sequences. The core sequence for ATOM-2 binding epitope is "His-Met-Asp-Ser-Pro-Asp-Leu-Gly-Thr ," which is present in all pET-expression vectors encoding S-tag fusion pro teins. Because the ATOM-2 binding region does not overlap with the S-protei n binding sequence, a convenient tool is provided for the simultaneous or a lternative detection, purification, and analysis of recombinant S-tagged pr oteins to conventional S-proteins.