Expression And Hydroxylamine Cleavage Of Thymosin Alpha 1 Concatemer And The Analysis Of Its Mechanicsim

Thymosinα1(Tα1) is an acidic protein composed of 28 amino acid residues with isoelectric point 4.2 and molecular weight 3108.As a potential immunologically active thymix polypeptide,it plays an important role in enhancing the immune system. 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 SARS,lupus erythematosus,cancers etc.Human thymosin alpha 1(Tα1) is an important peptide,and many studies have reported the expression of this peptide.In this study,we designed and synthesized the Tal gene according to the E.coli codon usage preference and constructed the 6×Tα1 concatemer gene.The latter was inserted into an E.coli expression vector pET-22b (+),thereby forming a recombinant plasmid that was subsequently transformed into E. coli BL21(DE3).After induction with 1 mM IPTG at 37℃for 7 h,the concatemer protein was successfully expressed in E.coli then cleaved by hydroxylamine,which could cleave the amino acid residues of Asn-Gly,particularly to release the Tα1 monomer.Urea-SDS-PAGE and mass spectrometry revealed that the recombinant protein was cleaved,as intended.The bioactivity of the Tα1 monomer was analyzed by an MTT assay.The results preliminarily showed that similar to the standard control Tα1,the monomer peptide could stimulate the proliferation of mice spleen lymphocytes and decrease the mitochondrial activity of HepG2 liver tumor cells and SPC-A-1 lung tumor cells.Reactive oxygen species(ROS) are constantly generated and eliminated in the biological system and play important roles in a variety of normal biochemical functions and abnormal pathological processes.The Tα1 loaded leukomonocytes,which were isolated from mice spleens,exhibited a higher reactive oxygen species(ROS) level and a lower reduced glutathione(GSH) level;however, HepG-2 cells treated with Tα1 exhibited lower ROS level and higher GSH level. When treated with Tα1,the population of leukomonocytes in the G₂ phase increased. However,in Tα1-treated HepG-2 cells,the cell cycle was delayed in the G₁ phase,and HepG-2 cell growth slowed due to dephosphorylation of the serine-threonine kinase Akt.This is the first report on 6×Tα1 concatemer expression and hydroxylamine cleavage of the concatemer protein to prepare the Tα1 monomer.This study provides the basis for the preparation of active Tα1 and provides an insight into the Tα1-induced signaling mechanisms in liver tumor cells and suggests the feasibility of using Tα1 as an antitumor agent.