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

• Autorzy: Ye M.H. , Chen J. L. , Zhao G. P. , Zheng M. Q. , Wen J.
• Języki publikacji: EN

Abstrakty

EN

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.

• Wydawca: -
• Czasopismo: Animal Science Papers and Reports
• Rocznik: 2013
• Tom: 31
• Numer: 1
• Opis fizyczny: p.63-71,fig.,ref.

Twórcy

autor

Ye M.H.

• 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
• College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China

autor

Chen J. L.

• 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

autor

Zhao G. P.

• 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

autor

Zheng M. Q.

• 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

autor

Wen J.

• 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

Bibliografia

• Averna M., De Tullio R., Capini P., Salamino F., Pontremoli S., Melloni E., 2003 − Changes in calpastatin localization and expression during calpain activation: a new mechanism for the regulation of intracellular Ca2+-dependent proteolysis. Cellular and Molecular Life Sciences 60,2669-2678.
• Byun S.O., Zhou H., Forrest R.H., Frampton C.M., Hickford J.G., 2008 − Association of the ovine calpastatin gene with birth weight and growth rate to weaning. Animal Genetics 39,572-573.
• Casas E., White S.N., Wheeler T.L., Shackelford S.D., Koohmaraie M., Riley D.G., Chase CC Jr., Johnson D.D., Smith T.P., 2006 − Effects of calpastatin and microcalpain markers in beef cattle on tenderness traits. Journal of Animal Science 84, 520-525.
• Chung H., Davis M., 2012 − Effects of genetic variants for the calpastatin gene on calpastatin activity and meat tenderness in Hanwoo (Korean cattle). Meat Science 90, 711-714.
• Enns D.L., Raastad T., Ugelstad I., Belcastro A.N., 2007 − Calpain/calpastatin activities and substrate depletion patterns during hindlimb unweighting and reweighting in skeletal muscle. European Journal of Applied Physiology 100, 445-455.
• Gandolfi G., Pomponio L., Ertbjerg P., Karlsson A.H., Nanni Costa L.,Lametsch R., Russo V., Davoli R., 2011 − Investigation on CAST, CAPN1 and CAPN3 porcine gene polymorphisms and expression in relation to post-mortem calpain activity in muscle and meat quality. Meat Science 88, 694-700.
• Kanazawa Y., Kaneshiro Y., Sawa M., Yasuoka H., Nojima T., Ohosone Y., Mimori T., 2000 − Domain reactivity of autoantibodies to calpastatin in patients with systemic rheumatic diseases. Modern Rheumatologyy 10, 38-44.
• Li S., Goldberg E., 2000 − A novel N-terminal domain directs membrane localization of mouse testis-specific calpastatin. Biology of Reproduction 63, 1594-1600.
• Lindholm -Perry A.K., Rohrer G.A., Holl J.W., Shackelford S.D., Wheeler T.L.,Koohmaraie M., Nonneman D., 2009 − Relationships among calpastatin single nucleotide polymorphisms, calpastatin expression and tenderness in pork longissimus. Animal Genetics 40,713-721.
• Molinari M., Carafoli E., 1997 − Calpain: a cytosolic proteinase active at the membranes.Journal of Membrane Biology 156, 1-8.
• Niu X., Guiltinan M. J., 1994 − DNA binding specificity of the wheat bZIP protein EmBP-1.Nucleic Acids Research 22, 4969-4978.
• Nonneman D., Lindholm -Perry A.K., Shackelford S.D., King D.A., Wheeler T.L., Rohrer G.A., Bierman C.D., Schneider J.F., Miller R.K., Zerby H., Moeller S.J., 2011 − Predictive markers in calpastatin for tenderness in commercial pig populations. Journal of Animal Science 89, 2663-2672.
• Schenkel F.S., Miller S.P., Jiang Z., Mandell I.B., Ye X., Li H., Wilton J.W., 2006 − Association of a single nucleotide polymorphism in the calpastatin gene with carcass and meat quality traits of beef cattle. Journal of Animal Science 84, 291-299.
• Takano J., Watanabe M., Hitomi K., Maki M., 2000 − Four types of calpastatin isoforms with distinct amino-terminal sequences are specified by alternative first exons and differentially expressed in mouse tissues. Journal of Biochemistry 128, 83-92.
• Tang W.H., Zhang J.L., Wang Z.Y., Hong M.M., 2000 − The cause of deviation made in determining the molecular weight of His-tag fusion proteins by SDS-PAGE. Acta Phytophysiologica Sinica 26, 64-68.
• Zhang Z. R., Jiang X. S., Du H. R., Zhu Q., Li X. C., Yang C. W., Liu Y. P., 2012 −Characterization of the expression profiles of calpastatin (CAST) gene in chicken. Molecular Biology Reports 39, 1839-1843.