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

International Journal of Molecular Sciences

Hong Xiao¹  Yawei Shi²  Jingming Yuan²  Yuming Huang² 

[1] 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

关键词: PICK1;    BARc;    human rhinovirus 3C protease cleavage;    ammonium sulfate precipitation;    inter-molecular interaction;   

DOI  :  doi:10.3390/ijms10010028

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).