Specific regions of core protein of phosphacan, one of the chondroitin sulfate proteoglycans, were expressed as fusion proteins with histidine-tag (His-tag) in Escherichia coli (E.coli) and were affinity purified using nickel-nitrilotriacetic acid (Ni-NTA) matrix. cDNA fragments encoding amino acid residues 343-446 (P3) and 1-340 (P4) of phosphacan core protein were amplified by polymerase chain reaction from E18 rat brain mRNA as template. The amplified products were subcloned into pQE30
vector and were introduced into E.coli strain M15 [pREP4] for the expression. The His-tagged fusion proteins were expressed by cultivating the transformants at 37℃ for 5h in the presence of 1mM IPTG. His-tagged P3 fusion protein (His-P3) was expressed as soluble form, and was purified
using Ni-NTA matrix. His-tagged P4 fusion protein (His-P4) which was sequestered into insoluble inclusion bodies was treated with 8.0M urea to solubilize, and then was purified under denaturing conditions.
core protein (コア蛋白)
recombinant protein (融合蛋白)