Expression,Purification, And Biological Activity Of Recombinant Human Thymosin β4

Background:Thymosin β4is an important actin chelating molecule in eukaryotic cells, it is widely distributed in most tissues and cells of the human body, and have a variety of biological activities. It participates in a variety of pathological and physiological processes, such as cell migration, angiogenesis and inflammation. In recent years, a lot of studies have shown that thymosin β4plays an important role in tissue repair, particularly, in skin wound healing.Objective:Clone and express the human thymosin β4protein in prokaryotic expression systems by using the gene recombination technology. And biological activities of the purified protein product were examined by both MTT methods in mice embryonic fibroblasts and its effects on a mouse-skin-wound-healing model. The purpose of present study was to set up a simplified in vitro technology for production of the thymosin β4protein with biological activity, which was confirmed for further study of its effects in skin wound healing.Methods:Using the gene recombination method to combine the human thymosin β4gene into the prokaryotic expression vector pTXB1, then the vector pTXB1was used to induce the expression of this fusion protein T04-intein-CBD in E.coli. After being purified by chitin beads, the expressed protein product is tested by HPLC, N-terminal sequencing and Western blot and other methods to identify the purifications. The biological activity of the recombinant human thymosin β4was tested in mice embryonic fibroblasts by MTT method and its effects on repairment of skin tissue damage was tested in mice skin-wound-healing model by skin histological examinations.Results:The recombination of the prokaryotic expression vector pTXB1-Tβ4was successfully constructed, and the fusion protein Tβ4-intein-CBD could be effectively expressed in E.coli BL21(DE3) under IPTG induction; after being purified by chitin beads and refined by S-100, we could get recombinant human thymosin β4protein product with high purity, which was identified of stable physical and chemical characters. The MTT results showed that recombinant human thymosin β4protein could promote the proliferation of3T3fibroblasts significantly (P <0.05). Results of histological and morphological studies on mice skin wound model showed that the recombinant human thymosin β4protein could promote epidermal cell migration, angiogenesis and collagen deposition, and promote hair follicle hyperplasia as well. Conclusion:By constructing recombinant prokaryotic expression vector pTXB1-TP4, expressing fusion protein Tβ4-intein-CBD in E.coli, and purifying the target protein by the Chitin beads, we set up a simplified and effective technology for the production of recombinant human thymus prime β4protein in vitro, the protein product has been confirmed of its biological activity, and the effectiveness on healing of skin wound.