Biosynthesis Of Thymosin β4 And Relative Investigations

Thymosin (β4 as a member ofβthymosins is a small protein containing 43aa and its N-terminal is acetylated. It has multiple significant physiological functions in tissue anagenesis, wound healing, and mudolating dynamic balance of acin, tumor invasion and transfer, cell apoptosis, inflammation, angiogenesis, hair follicle and so on. Some commercial companies such as RegeneRx have used chemically synthesized Tβ4 for its phaseⅡclinical treatment. As there are limitations of tissues extraction and chemical synthesis, people try to produce it in a biological way. But there are two bottlenecks to its biosynthesis:the difficulties in obtaining Nα-acetylation and expressing small peptides.In our study, a small Spl DnaX Intein (136aa) was choosed.The recombinant vector pET-Tβ4-Intein expressing the fusion protein was constructed by fusing the target protein and his tag to the N- and C-terminal of Spl DnaX Intein respectively, then it was co-transformed with acetyltransferase expressing plasmid pACYC-nat into E. coli BL21(DE3) cells. After being induced by lactose, Nα-acetylated Tβ4-intein was obtained. The splicing reaction of intein was blocked as the C-termini Asn was mutated to Ala, and N-terminal cleavage can be induced byβ-mercaptoethanol to release the target protein. The target protein was further purified by hydrophobic interaction, reversed phase, and positive ion exchange chromatographys. Then RP-HPLC, Q-TOF MS and Q-TOF MS/MS were employed to anlaysis the purity, identify the precise molecular weight and the site of acetylation respectively. These results suggested that the recombinat Tβ4 was fully Nα-acetylated and with native primary amino acid sequence. Three mutants of Tβ4 were prepared in the same way. The method is simple, robust and cost-effective, easy to scale up for biotechnology applications.61.79 mg target protein per liter culture was acquired and the purity was above 96.7% through the expression and purification processe. All of Tβ4 and its three mutants have the ability to bind actin. This study laid a foundation for further investigation of the function and the application of Tβ4.