Preparation And Bioactivity Evaluation Of Recombinant Human Thymosin Beta-4Expressed In Escherichia Coli

Thymosin beta-4is encoded by the TMSB4X gene in human beings. The protein in humans consists of43amino acids (msdkpdmaei ekfdksklkk tetqeknplp sketieqekq ages) with a molecular weight of4982Da. It has an isoelectric point of5.1and an acetylated serine residue at N terminal. The protein contains two a helixlocating between the4-16and30-40amino acid residues. Thymosin β4plays an important role in biomedicical process. It has been shown that thymosin β4has a variety of biological functions in cell adhesion and migration, tissue repair, angiogenesis, inflammation response and apoptosis. To get enough thymosin04, chemical extraction or synthesis acts as the main tools although it is difficult to obtain high purified thymosin (34by extraction or the high cost of chemical synthesis strictly limits its medical application. The analysis of cDNA and amino acid sequences of thymosin β4provides a way to get the peptide by genetic techniques. However, thymosin β4is too small to be stable in the host cells because of the protease degradation which neutralize the overexpression and restricts its industrialization.To overcome the above problems, a tandem repeat sequence encoding a triploid human thymosin β4spaced by a linker was designed and synthesized according to the codon usage bias of E. coli. The restriction endonucleases site of NcoI and XhoI was respectively introduced to the upstream and downstream of the gene. The recombinant triploid thymosin β4-coding gene fragment was recovered and linked with the prokaryotic expression vector pET22b(+) after NcoI and XhoI degradation. The ligation mixture was used to transform the competent cells of E. coli Top10and the recombinants detected by restriction enzyme digestion and sequence analysis was transformed into the expression host cells of E. coli BL21(DE3) and introduced by lactose to express the tandem repeat gene coding for triploid human thymosin β4. SDS-PAGE analysis showed that the recombinant human thymosin P4was expressed in BL21(DE3) as soluble fraction at a high level of28%in the total soluble protein. Most contaminant proteins could be removed from the cell lysate supernatant by boiling and high purified recombinant protein (92%) could be obtained through Ni affinity column chromatography. Rosette experiments, lymphocytes proliferation assay, cell migration assay and mouse hair regrowth experiments were employed to evaluate the bioactivity of the recombinant triploid human thymosin β4. The results showed that the recombinant triploid human thymosin β4could promote cell proliferation and migration and activate the hair follicle.