Prokaryotic expression, purification of chicken calpastatin protein and production of calpastatin polyclonal antibody
Journal Title: Animal Science Papers and Reports - Year 2013, Vol 31, Issue 1
Abstract
The open reading frame of chicken calpastatin (CAST) gene composed of 2,301 base pairs was ligated into a prokaryotic expression vector pET21a (+) to yield pET21a - CAST. The C-terminal His-tagged CAST protein was then expressed in E. coli. BL21 (DE3). SDS-PAGE analysis confirmed the successful expression of the fusion protein following induction with isopropyl-β-Dthiogalactopyranoside (IPTG). The recombinant protein consisted of 776 amino acid residues with an apparent molecular weight of approximately 110 kDa. It was primarily expressed as a soluble protein with a heat-stable feature. After being purified by Ni2+-NTA affinity resin, a polyclonal antibody was raised against the purified His-tagged CAST protein in rabbits. The reactivity and specificity of the polyclonal antibody were both subsequently characterized by ELISA. The study provides an important experimental tool for further research on the quantification of chicken CAST protein.
Authors and Affiliations
M. H. Ye, J. L. Chen, G. P. Zhao, M. Q. Zheng, J. Wen
Keywords
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