Expression And Purification Of Thymosin α1in Pichia Pastoris

Thymosin al(Tal), an acidic small molecule peptide, which composed of28amino acid was separated from calf thymus tissue TF5. The isoelectric point of natural Tal is4.2, with a acetylation at N-terminal, and the molecular mass is3.108KD. Its structure is simple without glycosylation and disulfide bond. As a biological response regulating factor, Tal is a kind of specific sign of T cells surface, and would help T cell mature and differentiate, it also can stimulate T cell, NK cell to secrete IL-2, a-interferon, and promote the generation of IL-2receptor. Tal is widely used in the immune suppression and immune adjustment disorder disease, it has been widely used in clinical. In addition, Tα1has significant therapeutic effect as a immune enhancer.At present, the bulk drug of Tα1is mainly for chemical synthesized, which is hard for the clinical application due to its high price, difficult to remove impurities formed in the synthesis process, pollution and some certain side effects. While the method of extracting from animal thymus tissue directly to get thymosin components is restricted by the raw material source. Due to the importance of Tal clinical application value, and its biological safety and effectiveness of drugs, more and more attention has been payed on the method of suitable genetic engineering to obtain Tal protein. So the amplification and industrialization has become one of the most popular researches. According to the reports, Tal has achieved expression in prokaryotic, eukaryotic and plant system.This research adopted the Pichia pastoris expression system which is suitable for expressing eukaryotic proteins to prepare Tal by genetic engineering strain fermentation. In order to construct the expression vector pPIC9K-Tα1, The artificial synthesized Tal monomer gene was amplified by primer X1and X2, and inserted into the pPIC9K plasmid with EcoRI and EcoRV by the way of a sticking end and a blunt end, then it was transformed into Pichia pastoris GS115to construct recombinant strain. Recombinant strain was induced by methanol and expression condition was optimized. Fermentation medium supernatant was purified by DEAE52and SephadexG-25to get Tal. The best inducing conditions of the recombinant strains were identified as:temperature was28℃, time was72h, methanol for the final concentration was1%, induced by pH6.0, and the best induction time for seed medium was18h. Tricine- SDS-PAGE and HPLC results indicated that Tal gene was expressed in Pichia pastoris successfully and the expression level was about4.8mg/L by the method of Bradford. The molecular weight was3.066KD by the mass spectrometry, consistenting with the theoretical one.