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)

Expression of Phosphacan, a chondroitin sulfate proteoglycan, core protein in Esherichia coli as a fusion protein with glutathione S-transferase

伊藤 昔子 岡山大学医療技術短期大学部衛生技術学科
岡本 基 岡山大学医療技術短期大学部衛生技術学科
抄録
Optimal conditions for expressing a specific region of core protein of phosphacan, a chondroitin sulfate proteoglycan known as receptor type protein tyrosine phosphatase, as fusion protein with glutathione S-transferase (GST) in E.coli were examined. DNA fragments inserted into the expression vector (pGEX-4T-1) were amplified by RT-PCR using mRNA purified from E18 rat brain as template. Primers attached with BamH I or EcoR I restriction site on 5' end were used to amplify first strand cDNA by PCR. Before ligation into the pGEX-4T-1 for GST fusion protein, PCR products were once cloned using T-A cloning system because they were not directly ligated into the pGEX-4T-1. E.coli strain BL21 was transformed by pGEX-4T-1 ligated with restriction DNA fragment cut out from pCR II plasmid vector of T-A clonig system. The growth of transformed BL21 was not different between the colony incubated at 37℃ for 24-48h and the colony stored at 4℃ for 7-10 days after 24h incubation at 37℃. The desirable OD(550) of culture medium for inducing the expression of fusion protein by isopropylthio-β-D-galactoside (IPTG) was from 0.6 to 1.0, because expression of native E.coli proteins per ml of culture medium was increased relatively when IPTG was added at OD(550) more than 1.0. The expression of fusion protein reached plateau around 6h after the induction. Relative expression of native E.coli proteins per ml of culture medium increased thereafter. Therefore, it may be desirable to purify the fusion protein around 6h after the induction.
キーワード
phosphacan (フォスファカン)
glutathione S-transferase (グルタチオン-S-トランスフェラーゼ)
BL21
IPTG
fusion protein (融合蛋白)
ISSN
0917-4494
NCID
AN10355371