title: Intracellular expression and purification of the Canstatin-N protein in Pichia pastoris.
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).
authors.affiliation: 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
authors.forename: Huixiang
authors.initials: H
authors.lastname: Yin
authors.forename: Zhenwang
authors.initials: Z
authors.lastname: Liu
authors.forename: Ailian
authors.initials: A
authors.lastname: Zhang
authors.forename: Tianyuan
authors.initials: T
authors.lastname: Zhang
authors.forename: Jinxian
authors.initials: J
authors.lastname: Luo
authors.forename: Jincheng
authors.initials: J
authors.lastname: Shen
authors.forename: Liping
authors.initials: L
authors.lastname: Chen
authors.forename: Bing
authors.initials: B
authors.lastname: Zhou
authors.forename: Xian
authors.initials: X
authors.lastname: Fu
authors.forename: Ceyi
authors.initials: C
authors.lastname: Fu
authors.forename: Zehua
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D020533:Angiogenesis Inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D002645:Chickens; D049033:Chorioallantoic Membrane / Q000378:metabolism; D024141:Collagen Type IV / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005285:Fermentation; D005822:Genetic Vectors; D061307:Human Umbilical Vein Endothelial Cells / Q000473:pathology*; D006801:Humans; D008297:Male; D008546:Melanoma, Experimental / Q000098:blood supply / Q000235:genetics / Q000517:prevention & control; D051379:Mice; D008807:Mice, Inbred BALB C; D018919:Neovascularization, Physiologic; D010843:Pichia / Q000254:growth & development* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; C502625:COL4A2 protein, human; D024141:Collagen Type IV; D011994:Recombinant Proteins