A comparison of phosphospecific affinity reagents reveals the utility of recombinant Forkhead-associated domains in recognizing phosphothreonine-containing peptides
| Autor: | Noman Shah, Oluwadamilola Bankole, Leon A. Venegas, Kritika Pershad, Brian K. Kay |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Models Molecular Phage display Bioengineering Biology Antibodies Article law.invention 03 medical and health sciences Protein Domains law Peptide Library Ca2+/calmodulin-dependent protein kinase Humans Protein Interaction Domains and Motifs Amino Acid Sequence Molecular Biology Transcription factor Genetics Kinase Affinity Labels Forkhead Transcription Factors General Medicine Recombinant Proteins 030104 developmental biology Phosphothreonine Biochemistry Polyclonal antibodies Recombinant DNA biology.protein Phosphorylation Peptides Biotechnology |
| Zdroj: | New biotechnology. 33(5 Pt) |
| ISSN: | 1876-4347 |
| Popis: | Phosphorylation is an important post-translational event that has a wide array of functional consequences. With advances in the ability of various technologies in revealing and mapping new phosphosites in proteins, it is equally important to develop affinity reagents that can monitor such post-translational modifications in eukaryotic cells. While monoclonal and polyclonal antibodies have been shown to be useful in assessing the phosphoproteome, we have expanded our efforts to exploit the Forkhead-associated 1 (FHA1) domain as scaffold for generating recombinant affinity reagents that recognize phosphothreonine-containing peptides. A phage display library of FHA1 variants was screened by affinity selection with 15 phosphothreonine-containing peptides corresponding to various human transcription factors and kinases, including human Myc, calmodulin-dependent protein kinase II (CaMKII), and extracellular-signal regulated kinases 1 and 2 (ERK1/2). The library yielded binding variants against 10 targets (66% success rate); success was largely determined by what residue occurred at the +3 position (C-terminal) to the pThr moiety (i.e., pT+3). The FHA domains binding Myc, CaMKII, and ERK1/2 were characterized and compared against commercially available antibodies. All FHA domains were shown to be phosphorylation-dependent and phosphothreonine-specific in their binding, unlike several commercial monoclonal and polyclonal antibodies. Both the pThr and the residue at the pT+3 position were major factors in defining the specificity of the FHA domains. |
| Databáze: | OpenAIRE |
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