The Biosynthesis Of Thymosinα1 And The Analysis Of Its Anti-tumor Mechanicsim

Human thymosin al (Tα1) is a biological polypeptide composed of 28 amino acid residues with immune-enhancing biological activity. It has widely functions such as enhancing the immune system, anti-tumor, endocrine regulation, and so on. At present, it is mainly used to cure chronic hepatitis B, hepatitis C clinically and as a powerful anti-virus drug for the treatment of bacterial or fungal infections, immunodeficiency, cancers, lupus erythematosus, etc. Thymosin alpha 1(Tα1) is so important, that it is meaningful to study either the expression or the function of this peptide. In this study, we transferred the Tal gene into E. coli to express it and make some chemically modification, and explored its role of tumor cell cycle arrest.First of all, we designed and synthesized the Tα1 gene according to the E. coli codon usage preference and constructed the with the intein self-shearing sequence concatemer gene (Ssp). We connected it to pET-32a and successfully constructed pET-32a-Ssp-Tal, transformed into E. coli BL21 (DE3) to make it expressed effectively (recombinant protein was above 50% of total protein). We confirmed that the target protein expressed in bacteria with soluble form and optimized the expression conditions (37℃, IPTG concentration is 0.5 mM), and through a series of processes (such as bacterial broken, centrifugal, Gel chromatography and Ni+ affinity chromatography), we purified the recombinant protein. We inducted it to self-shear through changing the pH and release the Tal monomer, then acetylated the Tal peptide with acetic oxide in vitro. HPLC separated the productions and Mass spectrometry revealed the weight of monomer was same with the nature Tal (3108 kD). In addition, we studied the expression levels of some cell cycle regulatory factors after HepG-2 cell be treated by Tal by western-blot, the results showed that Tα1 can reduce the Akt path and activate the p53 path, and can block the cell cycle by regulating the expression of cell cycle regulatory factors (cyclins and CDKs down while CDKI up) in the intracellular level. In this study, we built the fusion protein and expressed it in E. coli, got the Tal monomer by the intein self-shearing, so that the simple operations and the low costs which can be expected will make it possible to product Tal with large scale to meet clinical needs. We also found that Tα1 can regulate the expression level of some cell cycle regulators to inhibit tumor growth. And it provided an insight into the Tal-induced signaling mechanisms in liver tumor cells and suggests the feasibility of using Tα1 as an anti-tumor agent.