Cloning of Collagen VI COL6A2 and Its Expression in Pichia pastoris

The collagen Ⅵ chain α2 gene was amplified from human ollagen Ⅵ genomic DNA and subcloned into the vector of pPIC9K,and verified by DNA sequencing.The resultant recombinant plasmid pPIC9K-COL6A2 was digested by SalⅠ and transformed into the competent celss of Pichia pastoris strain GS115 through electroporation.Mut(methanol utilization)+ and Muts transformants were screened with MM and MD plates.Clony PCR was used to analyze Pichia pastoris integrants in order to determine if the gene of interest has been integrated into the Pichia pastoris genome.Cells with stable expression of collagen were screened in a medium containing G418.Regulated by α-factor,AOX1 gene promoter and termination signal of yeasts,the recombinant collagen was expressed and secreted from the cells.After induction with 1% methanol,SDS-PAGE analysis showed that the molecular weight of recombinant collagen was approximately 32 kD.