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

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 (700μg/ml). d-sorbitol was selected as the only carbon source. The fermentation was carried out in a 50L bioreactor at a 20L working volume. After 48h 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 780mg/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).