人源CDK2基因在大肠杆菌和毕赤酵母中的表达研究

he cyclin-dependent kinases (CDKs) are activated through binding of cell cycle proteins and then they drive the cell cycles. Among various CDKs, CDK2 plays a key role in the cell cycle regulation. In this study, we amplified CDK2 full-length gene by PCR and then cloned it into a modified bacterial expression vector pET28a (+)-GST-TEV as well as Pichia pastoris expression vector pPIC3.5K. Then, we screened for the positive recombinant plasmid pET28a-GST-TEV-CDK2 and transformed it into E.coli Rosetta before the protein expression was induced by IPTG; Meanwhile, the positive recombinant plasmid pPIC3.5K-CDK2 was transformed into Pichia pastoris GS115 and the expression was induced by methanol. We analyzed the protein expression by SDS -PAGE and Western-Blot. Also, the secondary structures of the proteins were studied by circular dichroism. The results showed that the two expression vectors were successfully constructed and both expression systems could carry out the expression of the target protein. Compared with that in E. coli, the expression of CDK2 in Pichia pastoris GS115 increased by 1.36mg/ml and stability increased by 50.3%.