Bulletin of Faculty of Health Sciences Okayama University Medical School
Published by Faculty of Health Sciences Okayama University Medical School

<Formerly known as>
岡山大学医療技術短期大学部紀要 (1巻-9巻)

Affinity purification of phosphacan core protein expressed in Escherichia coli as histidine-tagged fusion protein

Ito, Sekiko
Mori, Shuji
Abstract
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.
Keywords
phosphacan (ホスファカン)
core protein (コア蛋白)
His-tagged proteins
recombinant protein (融合蛋白)
ISSN
0917-4494
NCID
AN10355371