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

Gene
Hui-Xiang YinZe-Hua Zhang

Abstract

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 μg/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 A(600)=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).

References

Jan 8, 2000·The Journal of Biological Chemistry·G D KamphausR Kalluri
Dec 19, 2003·Biochemical and Biophysical Research Communications·Guo-An HeRui-Fang Li
Sep 22, 2006·Journal of Industrial Microbiology & Biotechnology·A L ZhangH L Li

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