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.