Cloning Of Type Vi Human Collagen Gene Col6A2 And Its Secreted Expression In Pichia Pastoris
Keywords
Collagen, Ppic9K, Pichia Pastoris
Abstract
The type VI collagen ¦Á2 chain gene was obtained using the PCR method using type VI collagen genomic DNA as a template, and the gene was inserted into the expression vector pPIC9K, and the target fragment contained in the recombinant plasmid pPIC9K-COL6A2 was sequenced in both directions. The correctly sequenced recombinant plasmid was linearized with restriction endonuclease SalI and transformed into Pichia pastoris GS115 competent cells by electroporation. The methanol utilization phenotype was screened by the MM/MD method, the integration of the target gene was identified by PCR and the G418 gradient screened high-copy transformants. Under the control of yeast ¦Á-Factor and AOX1 gene promoters and termination signals, the target protein is secreted and expressed extracellularly. In the fermentation broth induced by 1% methanol for 72 hours, the molecular mass of the recombinant protein was determined to be approximately 32kD by SDS-PAGE electrophoresis.
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Original research was done by Sun Yang, Xu Liqun, Wang Gang, Wang Hao, Chen Guang, Chen Huan
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