Expression And Purification Of Secreted Human Cyclophilin A In X-33 Pichia Pastoris Strain

Background:CypA was firstly extracted from bovine thymus in 1984. CypA consists of 165 amino acids, and its relative molecular weight is 18,000, which is widely expressed in the Cell Nucleus, Cytoplasm, and the Golgi Apparatus. CypA is the cellular receptor of CsA, the latter can be clinically used as the therapeutic agent in the organ transplants and autoimmune diseases. CypA can catalyze the peptide bond cis-trans isomerization and involves in signal transduction, regulates a variety of protein activity, plays the role of a molecular chaperone, assists other peptides with normal folding, assembling, transporting, and degradating, and plays an important physiological role in the process of DNA replication, transcription, cell signal transduction, and microtubule formation and repair.Inflammation and oxidative stress are the pathogenesis mechanisms of many diseases, especially for the cardiovascular diseases. The main marker of oxidative stress is the oxygen free radicals, among which reactive oxygen species (ROS) is most impotent. In the cardiovascular system, the process of inflammation and oxidative stress has been considered as an important factor of cardiovascular diseases like atherosclerosis, hypertension, restenosis after angioplasty. Currently antioxidant treatment on oxidative stress damage caused by cardiovascular disease has no obvious curative effect, which is mainly because of vitamin C and vitamin E and other antioxidants in cardiovascular tissues have no specificity. To explore the new target and mechanism for treatment of inflammation and oxidative stress mediated cardiovascular disease has an important significance. Recent studies have shown that CypA is a main SOXF, plays a key role in the pathogenesis of cardiovascular diseases, such as carotid artery injury, abdominal aortic aneurysm formation, atherosclerosis and myocardial remodeling. This study is mainly to construct pPICZaB-CypA expression vector, and convert it into Pythagorean yeast, induce its expression by methanol, then purify such protein and verify the biological functions of recombinant in order for further studies.Objective:1. Construct pPICZaB-CypA expression vector, and transformate it into X-33 yeast cells, induce its expression by methanol, and purify this protein.2. Evaluate the chemotactic effect of recombinant CypA on THP-1 cells in Transwell culture system.Methods:1、CypA cDNA containing the full-length CDS was obtained using one-step RT-PCR amplification, then construct pPICZaB-CypA expression vector, and verify its correctness by restriction enzyme digestion and gene sequencing.2、Transformate the pPICZaB-CypA vector into X-33 yeast cells, induce its expression by methanol, and detect its expression with SDS-PAGE.3、Purify CypA protein by cation exchange method, and detect its expression with SDS-PAGE detection.4、Verify the chemotaxis of purified CypA protein on THP-1 cells by Transwell method.Results:1、Restriction enzyme digestion and gene sequencing indicated the recombinant expression vector is correct.2、CypA was highly secreted from X-33 yeast cells after transfection.3、CypA protein was successfully purified with the DEAE cation exchange.4、the functional property of purified CypA was confirmed with method of the chemotaxis on THP-1 cells.Conclusion:Functional CypA can be obtained from X-33 yeast cells transformed with pPICZaB-CypA.