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Animal Science Papers and Reports

Prokaryotic expression, purification of chicken calpastatin protein and production of calpastatin polyclonal antibody

M. H. Ye
The Key Laboratory for Farm Animal Genetic Resources and Utilization of the Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100094, China
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-D-
thiogalactopyranoside (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 Ni
2+
-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

Keywords :
calpastatin / chicken / gene / polyclonal antibody / prokaryotic expression

Date Deposited : 02 Apr 2015 09:43

Last Modified : 02 Apr 2015 09:43

Official URL: http://www.ighz.edu.pl/?p0=5&p1=34

Volume 31, Number 1, - 2013

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