Cloning The Cyclophilin Gene Of Yarrowia Lipolytica Followed By Constructing The Targeting Vector

Cyclophilin (CyP) belongs to a family of highly conserved and multifunctional protein that is found in both prokaryotes and eukaryotes. CyP possesses peptidyl-prolyl cis-trans isomerase(PPIase) activity which can catalyze the interconvertion of the cis and trans isomers of the peptidyl-prolyl bonds in peptide and affect the process of protein folding and assembling. In addition, CyP can work as molecule chaperones and plays roles in signalling pathways regulation under stress condition and RNA splicing. Since the initial discovery of CyP that is the intracellular target of the immunosuppressant drug cyclosporin A (CsA), about 130 CyPs isoforms have been identified in different organisms from bacteria to mammal. All members of CyP family have different molecular weight, distinct location and varying function. Yarrowia lipolytica is a "non-conventional" species of yeast, which was widely researched. Yarrowia lipolytica can use various lipids or proteins as carbon sources to produce methane. It also can be used to produce citric acid from alkanes, vegetable oils or glucose under aerobic conditions. Yarrowia lipolytica is routinely isolated from different food media (cheese, sausages) or from natural environments like oil fields. It is harmless for people.One survey has indicated that Yarrowia lipolytica has 13 putative cyclophilin genes, and little is known about the function of these genes; they are generally not linked to each other in the genome. One of 13 putative CyP gene was selected as the research object in Yarrowia lipolytica. Bioinformatic analysis of the CyP indicated that it contains 638 amino acid residues. The molecular weight is about 71 Kda, and the theoretical pI is 5.59. The CyP has been predicted by PSORT to be cytoplasmic, but this has yet to be confirmed practically. BLAST searching was performed on the CyP and we found that its similarity with the CyP A of H. sapiens is only 44.8 %, but those crucial amino acid residues which required for forming hydrophobic core and the roles, are highly conserved. It implies that the CyP family probably has a commonβ-barrel motif as its molecular core even though the overall sequence homology may be as low as 43%. In addition, structure analysis show that the 3-dimensional of CyP between Yarrowia lipolytica and H. sapiens by SwissModel. RT-PCR analysis showed that the result of the expression of CyP mRNA was identical to the expected results.The construction of the targeting gene vector was successfully completed, which will lay the foundation for the further researching of the function of the CyP gene.