Study On The Expression And Purification Of Recombinant Thymosin α1

Thymosin 1(Tα1) is a acidic protein composed of 28 amino acid residues with isoelectric point 4.2 and molecular weight 3102.Its amino acid sequence is: N-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu -Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-C。 T1 is a T-cell potentiating factor. It affects the maturation, differentiation and function of T-cells 。It can enhance the production of cytokines such as IL-2 and IFN- by mature T-cells and NK cells and up-regulate the expression of IL-2 receptors. Besides,it has been found that T1 can antagonize CD3-induced and glucocorticoid-induced thymocyte apoptosis,stimulate endothelial cell migration, angiogenesis and wound repair, reduce erythrocyte lipid levels and enhance the fertilizing capacity of human sperm cell .T1 is now widely used in clinic for treatment of infectious diseases including hepatitis B, C, HIV, maliganant tumors.and other immune defiency diseases.T1 was first isolated from a partially purified extract from calf thymus tissue termed thymosin Fraction 5 .At present,There are two ways of preperation of thymosin 1: One way is to extract from bovine thymus tissue , and the other is solid-phase synthesis.The first method is limited to its sources,and usually a mixture of thymosin peptides is available.As for synthetic T1,it is high-cost and expensive .We have carried out the study on T1 production by genetic engineering.First, T1 gene was cloned into the fusion expression vector pGEX-4XT-1. The recombinant plasmid was transformed into E.coli DE3(lys) and high-level expression of GST-T1 fusion protein was obtained.To produce a large amount of target protein,batch culture and fed-batch culture were carried out in a NBS-MICROS15L.T.D.R fermentor.In batch culture, the final cell density OD(600) was 5 and the amount of GST-T1 fusion protein was 56mg/L.Fed-batch culture was carrid out by means of controlling medium supplement andincreasing dissolved oxygen with higher gas flow rate. The final cell density and expression of GST-T1 rised up to 22 and 0.26g/L respectively.The fusion protein was soluble and over 30% of the total protein in the supertanant after ultrasonication .GST-T1 fusion protein was purified by affinity chromatography with a purity of over 95%. T1 was released after thrombin cleavage of the fusion protein . GST protein was removed with affinity chromatography,and after that T1 was purified by ion exchange chromatography.The purity of the final product was over 90%.Biological activity assay showed that purified T1 can enhance E-rossete formation of human PBMC(P<0.01) and stimulate the proliferation of mice spleen cells at the concentration of 10-7,10-8,10-9,10-10mol/L(P<0.01).The biological activity of purified T1 is similar to imported synthetic T1. To some extent ,we have solved the difficulties in the expression of low molecular weight peptides.The result of fermentation is stable and the purification method is simple and easy to scale-up.So this study will has some significance for large-scale production of T1 in the future...