Cloning, Expression, Purification And Identification Of Human Thymosinβ4 In E Coli

Thymosinβ4,firstly isolated form calf thymus by Low and his colleague in 1981, is one of the effective components of thymosin.Thymosinβ4 is evolutionarily conserved and composed of 43 amino acids with acetylserine at the NH₂ terminus.It has isoelectirc point of 5.1.Thymosinβ4 was involved in the development and differentiation of thymus-derived lymphocyte.Thymosinβ4 existed ubiquitously in vertebrates and was detected in blood,body fluid,wound fluid and expressed highly in macrophagocyte,platelet.A lot of evidences proved that thymosinβ4 could induce the differentiation of vessel endothelial cells;enhance angiogenesis;and promote wound healing and myocardiocyte cell survival and coronary vessel formation in myocardic infarction.Thymosinβ4 has a broad perspective in the treatment of skin, corneal wound,cardiac infarction,tumor,inflammation and some potential clinical application has entered clinical trial.However,currently thymosinβ4 used for research is synthesized through a prohibitively expensive chemical process.In this study,we explored the other avenue using genetic engineer method to obtain sufficient thymosinβ4 more economically. (一)Cloning,Expression and purification of human thymosinβ4 in E coli.At first,human thymosinβgene sequence optimized using E coli preferred codon to facilitate the expression in E coli.The whole gene was synthesized chemically by Bioasia corperation.The amplified gene,introduced two restriction enzyme site(Ndeâ… /Xhoâ… ) at the two end by PCR,was cloned into the pGEM-T vector.Then the thymosinβ4 gene was subcloned into the multiclonal site of pTXB1 vector and the plasmid was transformed into E coli ER2566.IPTG induces expression for 2 hours.The induced fusion protein was released from E coli by the method of ultrasonication.By affinity purification and DTT-induced self cleavage on column, untrafiltration and desaltation,recombinant human thymosinβ4 was got.The purity of recombinant thymosinβ4 ia above 90%by Tricine-SDS-PAGE analysis.(二)Biophyiscal characterization of recombinant thymosinβ4By Edman degradation method,N-terminal sequencing results showed the first five residues of recombinant protein were identical to the counterpart of the human thymosinβ4 derived from its cDNA sequence(SDKPD).The purified protein sample was analyzed by MALDI-TOF-MS using Applied Biosystems(ABI) 4800 MALDI-TOF mass spectrometry.The analysis result showed that the molecular mass determined for recombinant protein is 5394.90,which is compatible with the caculated value of 5394.95.After trypsin digestion,the recombinant protein was subject to peptide-mass-footprint(PMF) analysis.The results showed that recombinant protein had a good match with native thymosinβ4.All these results furtherly comfirm that recombinant protein is human thymosinβ4.(三)Biological activity of recombinant human thymosinβ4The biological activity of recombinant and synthetic human TB4 was evaluated by its promotion of T lymphocyte proliferation and differentiation as described previously with some modification.The lymphocyte proliferation assay was performed as previously described.Briefly,the spleen was aseptically removed from a BALB/c mouse.A suspension of single spleen cells was prepared by lysing the red blood cells and collecting into cell culture media(CCM) that contained RPMI1640 (Gibco),2mM glutamine,10%fetal bovine serum(Hyclone),and 100μg/ml of penicillin and streptomycin.Triplicate cultures were grown in 96-well flat-bottomed tissue culture plates at 5×105 cells/well.For the activity assay,recombinant or synthetic human TB4 was added to the cell suspension at a final concentration of 2-10μg/ml and afterwards the splenocytes were incubated for 6h in the presence of ConA(4μg/ml) at 37C in a humidified incubator with 5%CO₂ and 95%air. Splenocytes were cultured for 72h.Then activity assay was performed using CellTiter aqueous one solution cell proliferation assay(MTS) kit(Promega,USA)according to the manufacturer's instructions.Compared to chemically synthesized ones, recombinant human thymosinβ4 has similar activities in promotin proliferation and differentiation of lymphocyte.(å››)Recombinant human thymosinβ4 promote full-cutaneous wound healingUsing full-cutaneous wound model in Balb/c mouse,recombinant human thymosinβ4's effect on wound healing was evaluated.Briefly,Four full-thickness 3 mm punch wounds were made on the dorsal surface of each mouse.Recombinant human TB4 was applied topically to two of the wounds on each mouse(5μg in 50μl) at the time of wounding and again after 48 h.Controls for the TB4-topical treatment(the two remaining wounds on each mouse) received identical amounts of saline at the time of wounding and at 48 h.Mice were not bandaged.The experiment was terminated on day 7 post wounding.And then those mouse were subject to histological analysis.As indicated by the results of histological analysis,topically treatment with recombinant human thymosinβ4 can results in increased re-epithelialization,collagen deposition,and angiogenesis;and thus recombinant human thymosinβ4 has the ability of promoting full-cutaneous wound healing.