Intracellular expression and purification of the Canstatin-N protein in Pichia pastoris

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• 作者：Huixiang Yin^a ; ^{yinhxg@163.com} ; Zhenwang Liu^a ; ^{Izwang163@163.com} ; Ailian Zhang^a ; ^{Zhangailian6@yahoo.com.cn} ; Tianyuan Zhang^d ; ^{tianyuanzhang2005@yahoo.com.cn} ; Jinxian Luo^d ; ^{jinxianluo@yahoo.com} ; Jincheng Shen^a ; ^{oyanxuelae@163.com} ; Liping Chen^a ; ^{chenliping85@sina.com} ; Bing Zhou^b ; ^{544958195@qq.com} ; Xian Fu^a ; ^{LBSFX8@Sina.com} ; Ceyi Fu^c ; ^{Xf_vicar@yahoo.cn} ; Zehua Zhang^a ; ^{zj408694019@163.com}
• 关键词：SDS-PAGE ; dodecyl sulfate ; sodium salt-Polyacrylamide gel electrophoresis ; DEME ; Dulbecco's Modified Eagle Media ; bFGF ; basic fibroblast growth factor
• 刊名：Gene
• 出版年：2012
• 出版时间：1 August, 2012
• 年：2012
• 卷：504
• 期：1
• 页码：122-126
• 全文大小：678 K

文摘

Canstatin-N DNA fragment amplified from human genome was inserted into the MCS of pGAP9K*, an intracellular expression vector of Pichia pastoris, to generate pGAP9K*-can-N which was then transformed into P. pastoris GS115 by electroporation. A transformant was chosen as an engineering strain from the plate containing G418 (700 ¦Ìg/ml). d-sorbitol was selected as the only carbon source. The fermentation was carried out in a 50 L bioreactor at a 20 L working volume. After 48 h fermentation with continuous feeding of 25 % (w/v) d-sorbitol and 0.8 % PTM4, the cell grew to A₆₀₀ = 178 and intracellularly expressed Canstatin-N reached 780 mg/L. Snail enzyme was combined with water to crack P. pastoris and to release intracellular proteins. The purified recombinant Canstatin-N inhibited CAM angiogenesis and induced significant apoptosis of the human umbilical vein endothelial cell (EVC340).