Over-expression, rapid preparation and some properties of c-terminal BARc region in PICK1.
A DNA fragment encoding C-terminal BARc region (amino acids 128-416) of rat PICK1 (NP_445912 ) was inserted into a modified vector pMAL-s involving human rhinovirus 3C protease cleavage site to produce a recombinant plasmid, pMAL-s-barc. The construct can express the fusion protein, MBP-BARc in the soluble form in E.coli. To remove the MBP tag, MBP-BARc purified from amylose beads was digested with human rhinovirus 3C protease and the cleavage efficiency is about 95% when the ratio of protein / enzyme (w/w) reaches 50:1, as analyzed on SDS-PAGE. The enzymatic reaction mixture was rapidly separated into two parts, MBP in the supernatant and BARc in the precipitate at the concentration of 1 M ammonium sulfate. In such case, the target protein BARc could be economically produced in a soluble state to be as the sample for measuring its biochemical function, for example, protein-protein interaction and protein-lipid combination.
Xiao
Hong
H
Department of Pathology, The First Affiliated Hospital, Shanxi Medical University, Taiyuan 030001, P.R. China.|Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Institute of Biotechnology, Shanxi University, Taiyuan 030006, P.R. China.
Shi
Yawei
Y
Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Institute of Biotechnology, Shanxi University, Taiyuan 030006, P.R. China.
Yuan
Jingming
J
Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Institute of Biotechnology, Shanxi University, Taiyuan 030006, P.R. China.
Huang
Yuming
Y
Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Institute of Biotechnology, Shanxi University, Taiyuan 030006, P.R. China.
Wang
Junhua
J
Jiangsu Kingsley Group Co., Nanjing 210000, P.R. China.