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Int. J. Mol. Sci. 2009, 10(1), 28-36; doi:10.3390/ijms10010028
Article

Over-expression, Rapid Preparation and Some Properties of C-terminal BARc Region in PICK1

1, 2,#,* , 2,#, 2,* , 2 and 3
1 Department of Pathology, The First Affiliated Hospital, Shanxi Medical University, Taiyuan 030001, P.R. China 2 Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Institute of Biotechnology, Shanxi University, Taiyuan 030006, P.R. China 3 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 / Revised: 16 December 2008 / Accepted: 18 December 2008 / Published: 27 December 2008
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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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 PICK1; BARc; human rhinovirus 3C protease cleavage; ammonium sulfate precipitation; inter-molecular interaction
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).
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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.

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