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

• Published in: Gene Vol. 504; no. 1; pp. 122 - 126
• Main Authors: Yin, Huixiang, Liu, Zhenwang, Zhang, Ailian, Zhang, Tianyuan, Luo, Jinxian, Shen, Jincheng, Chen, Liping, Zhou, Bing, Fu, Xian, Fu, Ceyi, Zhang, Zehua
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2012
• Subjects: Angiogenesis; Angiogenesis Inhibitors - genetics; Angiogenesis Inhibitors - isolation & purification; Angiogenesis Inhibitors - metabolism; Animals; Apoptosis; Bioactivity; Bioreactors; Canstatin-N gene; carbon; Carbon sources; Cells, Cultured; Chickens; Chorioallantoic Membrane - metabolism; Collagen Type IV - genetics; Collagen Type IV - isolation & purification; Collagen Type IV - metabolism; continuous fermentation; D-Sorbitol; DNA; Electroporation; Endothelial cells; Enzymes; Expression vectors; Feeding; Fermentation; Genetic Vectors; genome; Genomes; Human Umbilical Vein Endothelial Cells - pathology; Humans; Intracellular expression; Male; Melanoma, Experimental - blood supply; Melanoma, Experimental - genetics; Melanoma, Experimental - prevention & control; Mice; Mice, Inbred BALB C; Neovascularization, Physiologic; Pichia - growth & development; Pichia - metabolism; Pichia pastoris; protein purification; proteins; Purification; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; sorbitol; umbilical vein; SDS-PAGE; Purification; Canstatin-N gene; DEME; Intracellular expression; Bioactivity; Pichia pastoris; bFGF
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2012.04.073

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). ► 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.