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 th...
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Published in | Gene Vol. 504; no. 1; pp. 122 - 126 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Netherlands
Elsevier B.V
01.08.2012
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | http://dx.doi.org/10.1016/j.gene.2012.04.073 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Article-2 ObjectType-Feature-1 |
ISSN: | 0378-1119 1879-0038 |
DOI: | 10.1016/j.gene.2012.04.073 |