Expression, Purification And Activity Detection Of Human Superoxide Dismutase - Thymosin α1 In Pichia Pastoris

Thyαl(Thymosin alpha-1), an small active peptide, plays an important role in improving immune systems and stimulating the functioning of lymphocyte. It also has been proved both in vitro and vivo that thyal can enhance the anti-virus, anti-mycobacteria and anti-fungus abilities of human body. It has been used as immunomodulator and biological response modifier in treating hepatitis, AIDS, infection, allergic reaction, autoimmune disease, cancer disease and recovery of immune system. Thyal can be synthesized in three ways currently:extractingfrom animal tissue, chemical synthesis and genetic synthesis. Most of the thyal in clinical application are extractedfrom animal tissue, which is very expensive and difficult to control the quality. Genetic synthesis of thyα1has the advantage of low cost and high quality. However, it has difficulties in synthesizing techniques due to its short molecular length.SOD(Superoxide dismutase) catalyze the reaction of reducing O2-·into H2O2and O2. It can protect living bodies from oxidative damage and decrepitude. SOD is very stable under different pH and temperatures. Its safty in vivo has been tested under variety of clinical experiments. It is nonpoisonous for human body except rare case of hypersensitivity. Therefore, SOD and its genetic derivativeshave been widely used in food, health products, cosmetics and drug industry.It is believed that SOD can trigger thesynergic effectwhich could enhance the activity of thyal. To improve the stability and immunization activity of thyal, thyal encoding gene, thya1, was fused to hSOD by using (G4S)n(n=1-2) as linker and expressed in Pichia pastorisin the present study. The recombinant fusion proteins were purified by Ni-NTA Resin Chromatography. The purity of the fusion protein is over80%based on the SDS-PAGE and BandScan analysis. The purified6His-hSOD-(G4S)i-thyal and6His-hSOD-(G4S)2-thyal respectively exhibited hSOD activity of120998U/mg and31279U/mg, and the E-rosette formation rate of38.4%and36.6%at a protein concentration of1×10-7mol/L. The results reported here suggest that the presence of G4S linker between two individual proteins can efficiently keep the structure of each of fused proteins. The fusion proteins keep both high SOD activity and high Thyal activity. Cellulose is the richest natural resource on our planet. It can provide us high-valued and reproducible biofuel which can not be used up. Xylanase is one of the cellulases that coulddegrade cellulose into biofuel. As its significantapplication in paper-bleaching, feedadditives and biofuel, xylanase has been widely studied recently. For better use in industry, itis yet to be improved and modified by genetic engineering methods due to its limitations on specific temperature rangeandpH range.Xylanases of family11xylanase from different sources have been cloned and expressed, and the crystal structures also have been determined to study their natures. There are already some researches on improvementof the enzyme’s thermostability. You Chun reported apoint-mutation G201C taken place in protein interior hydrophobic region in a family11xylanase (XynC) from Neocallimastix patriciarum, which caused greatly increased thermostability than the wild-type. The thermostability of the variant is correlation with hydrophobicity of the residues in site50and201. Further study indicates the mutant of XynC-G201, C60A-G201C, is even better in thermostability.We identified8mutants based on C60A-G201C. In this study, the mutants have been expressed and purified by Ni-NTA Resin Chromatography. Through inactivation rate determination, we found mutant37have higher ki than C60A-G201C.The far-ultraviolet circular dichroism signal showed that the Tm of the mutant37is is6.12℃higher than C60A-G201C, which also suggests higher. We built a3D model of mutant 37by homology modeling. The structure showed the mutant has a single point-mutationat site20, from Lys to Glu, and there is a hydrogen bond between Glu and Tyr in site20and29, which might be the reason of higher thermostabilityof the mutant.