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Over-expression, Rapid Preparation and Some Properties of C-terminal BARc Region in PICK1
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
Jiangsu Kingsley Group Co., Nanjing 210000, P.R. China
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed.
Received: 19 October 2008; in revised form: 16 December 2008 / Accepted: 18 December 2008 / Published: 27 December 2008
Abstract: 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.
Keywords: PICK1; BARc; human rhinovirus 3C protease cleavage; ammonium sulfate precipitation; inter-molecular interaction
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Xiao, H.; Shi, Y.; Yuan, J.; Huang, Y.; Wang, J. Over-expression, Rapid Preparation and Some Properties of C-terminal BARc Region in PICK1. Int. J. Mol. Sci. 2009, 10, 28-36.
Xiao H, Shi Y, Yuan J, Huang Y, Wang J. Over-expression, Rapid Preparation and Some Properties of C-terminal BARc Region in PICK1. International Journal of Molecular Sciences. 2009; 10(1):28-36.
Xiao, Hong; Shi, Yawei; Yuan, Jingming; Huang, Yuming; Wang, Junhua. 2009. "Over-expression, Rapid Preparation and Some Properties of C-terminal BARc Region in PICK1." Int. J. Mol. Sci. 10, no. 1: 28-36.