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 together with the 103 amino acid large S-protein the whole ribonuclease S-protein. Its small size and excessive solubility have made the S-tag an excellent fusion partner in the production of recombinant proteins, and a large variety 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 recombinant proteins, and in this context, a novel MAb termed ATOM-2 was generated that specifically binds S-tagged proteins, which have been expressed using pET-vectors. Antigen specificity of ATOM-2 was confirmed in Western blot and enzyme-linked immunoadsorbent assay analysis, and using a series of amino acid deletion mutants, the binding epitope of ATOM-2 was precisely mapped. 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 proteins. Because the ATOM-2 binding region does not overlap with the S-protein binding sequence, a convenient tool is provided for the simultaneous or alternative detection, purification, and analysis of recombinant S-tagged proteins to conventional S-proteins.
Park
J H
JH
Protein Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejon.
Na
S Y
SY
Lee
H H
HH
Lee
Y J
YJ
Kim
K L
KL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes; D005822:Genetic Vectors; D020738:Leptin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology*; D051381:Rats; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000302:isolation & purification*; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D020738:Leptin; D015335:Molecular Probes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins; C060871:ribonuclease A S-peptide; D012259:Ribonuclease, Pancreatic