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.
Highlights
PICK1 is a protein kinase C (PKC) binding protein, initially identified by a yeast two-hybrid system [1]
We constructed a new vector pMAL-s, which is modified from pMAL- p2X by substituting Factor Xa cleavage site with human rhinovirus 3C protease cleavage site
Each reconstruct could more and less express the corresponding protein MBP-BARc, all of them existed in the precipitate except pMAL-s-barc in E.coli
Summary
PICK1 (protein interacting with C kinase 1) is a protein kinase C (PKC) binding protein, initially identified by a yeast two-hybrid system [1] It is a cytosolic protein containing a PDZ [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] domain at N-terminus as well as a BAR (Bin/amphysin/Rvs) domain and an acidic amino acid region close to the C-terminus [2]. In order to explore the biological function of BAR domain in rat PICK1 (NP_445912), we prepared a soluble fusion protein MBP-BARc and obtained the target protein BARc (amino acids 128-416). We constructed a new vector pMAL-s, which is modified from pMAL- p2X by substituting Factor Xa cleavage site with human rhinovirus 3C protease cleavage site In such case, the expression product, MBP-BARc could be cleaved into MBP and BARc by the protease. The finding provides a convenience for further studying the biological function of the BARc [9]
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