Prokaryotic Expression And Purification Of Purification Of Cyclophilin From Yarrowia Liplytica

Cyclophilins comprise a protein family of highly conservative structure which exist in almost all organisms. As the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilins can catalyze the cis-trans isomerization of the X-Pro peptide bonds. In the same time, cyclophilins work as molecular chaperones in helping proteins flod, assemble and transport. They are also playing pivotal roles in cyclosporin A-mediated immunosuppression and other events like programmed cell deaths, oxidative stress, cholesterol metabolism. The yeast Yarrowia lipolylica belongs to the ascomycetous yeast. It is an important industrial yeast which can survive wild range of pH conditions and take ordinary and cheap substances like glucose, fatty acids, hydrocarbons as substrates. Since Yarrowia lipolytica is nonpathogenic, it can be used in the food and medicine production. Yarrowia lipolylica secrets large amounts of metabolites like organic acids and proteins, so it was developed into a new yeast expression system. More than one hundred of cyclophilins have been identified, and there are also 13 putative cyclophilins through out the Yarrowia lipolytica genome.In this study, we picked one of the putative Yarrowia lipolytica cyclophilins (Gene ID:2909417) as our research object and obtained the gene information from the NCBI database. Primers were designed on the open reading frame of the cyclophilin gene. And the genomic DNA was extracted for amplification of the cyclophilin gene by PCR. TA cloning and sequencing was conducted to identify the insert, after which the expressive vector PET32a-CyP was constructed. Then the constructed expressive vector pET32a-CyP was transformed to E. coli BL21 (DE3) plysS for expression under induction of IPTG. The cells were breaked by ultrasonic. Then the induced cyclophilin protein was obtained by immoblized metal-chelate affinity chroma to graphy and identified by SDS-PAGE.Sequencing result confirmed that the full length cyclophilin gene has been cloned. Colony PCR analysis proved that expressive vector pET32a-CyP was correctly constructed. SDS-PAGE showed that The molecular weight of the induced cyclophilin protein was about 95KD. Conclusion:The successful prokaryotic expression of the Yarrowia lipolytica cyclophilin gene may lay the foundation for future structural and functional Studies.