1. Intracellular expression and purification of the Canstatin-N protein in Pichia pastoris
- Author
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Jincheng Shen, Tianyuan Zhang, Huixiang Yin, Ailian Zhang, Bing Zhou, Ce-Yi Fu, Zhenwang Liu, Fu Xian, Jinxian Luo, Zehua Zhang, and Liping Chen
- Subjects
Collagen Type IV ,Male ,Genetic Vectors ,Melanoma, Experimental ,Neovascularization, Physiologic ,Angiogenesis Inhibitors ,Apoptosis ,Chorioallantoic Membrane ,Pichia ,Pichia pastoris ,law.invention ,Mice ,law ,Genetics ,Human Umbilical Vein Endothelial Cells ,Animals ,Humans ,Cells, Cultured ,chemistry.chemical_classification ,Mice, Inbred BALB C ,Expression vector ,biology ,Electroporation ,General Medicine ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,Enzyme ,Biochemistry ,chemistry ,Fermentation ,Recombinant DNA ,Human umbilical vein endothelial cell ,Chickens ,Intracellular - 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).
- Published
- 2011