Intracellular expression and purification of the Canstatin-N protein in Pichia pastoris |
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Authors: | Yin Huixiang Liu Zhenwang Zhang Ailian Zhang Tianyuan Luo Jinxian Shen Jincheng Chen Liping Zhou Bing Fu Xian Fu Ceyi Zhang Zehua |
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Institution: | Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Haikou Hainan 571101, China. yinhxg@163.com |
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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). |
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Keywords: | SDS-PAGE dodecyl sulfate sodium salt-Polyacrylamide gel electrophoresis DEME Dulbecco's Modified Eagle Media bFGF basic fibroblast growth factor |
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