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 (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 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).

► Cloning of canstatin-N gene and constructing of engineering P. pastoris strain.
► Intracellular expression of 780 mg/L Canstatin-N in P. pastoris.
► Invention of snail enzyme combined water to crack P. pastoris for purification.
► Research of carbon sources for P. pastoris pGAP expression system.
► Activity analysis of the P. pastoris intracellular expressed Canstatin-N.