Expression, Seperation And Purification Of Recombinant Human Thymosin α1 In E.coli

Human thymosinα1(Tα1) , a peptide consisting of 28 amino acids withan isoelectric point of 4．2 and a molecular weight of 3 108 Da, is secreted byhuman thymus gland. As a potential immunologically active thymixpolypeptide, it plays a great role in enhancing the immune system. Atpresent, it is mainly used to cure chronic hepatitis B, hepatitis C clinically andas a powerful antivirus drug for treatment of SARS, lupus erythematosus,cancers etc. with good clinical effect.Considering the feasibility of producing human thymosinα1 in largescale, an over-expression vector had been constructed in our lab based on theplasmid pET28a(+). The first step was to choose the prefered codon of E. colifor synthesis recombinant human thymosinα1 (rhTα1) gene. The second stepwas to construct the fusion expression vector pET-rhTα1．Finally, the fusionexpression vector pET-rhTα1 was transferred into E.coli BL21 (DE3) to get ahigh-level expression of rhTα1．In order to prepare recombinant fusion proteinwith the transgenic E.coli, the optimal process for the target protein expression and purification was studied in detail. The optimal experiment conditions forthe genetic engineered E.coli were as follows: As genetically engineeredbacteria entered into recession period after a fermention of 14 hours,fermentation was terminated at 14 h post; Composition of culture medium:Glucose 6 g/L, tryptone 15 g/L, yeast extract 10 g/L, NaCl 10 g/L, K₂HPO₄ 11g/L, KH₂PO₄ 4．6 g/L, NHCl 2．20 g/L, MgSO₄ 1．1 g/L, and FeSO₄ 45 mg/L;Inoculation quantity was 10% of the culture medium; The induction wasstarted at its ogarithm growth metaphase and lasted for 6 h with IPTG 1．0mmol/L.The recombinant protein expressed in E.coli was usually accumulated asinclusion bodies in the bacterium. Therefore, how to refold proteins correctlyat high concentration with high yield was one of the critical techniques forrecombinant proteins production. Protein refolding by dilution was a moresimply method for protein renaturation than others. In our study, the crudeinclusin bodies were washed with 2 mol/L urea before refolding in order toremove impurities. Then the inclusion bodies were redissolved by 7 mol/Lguanidine hydrochloride containing 10 mmol/L of DTT. After that, thedenatured liquid was added to the renaturation solution by a pulse way at 0℃.For purification of target proteins, three types of chromatographs includingDEAE-Sepharose Fast Flow, Ni-chelating Sepharose and Sephadex G-25column were step by step run, and the rhTα1 purify obtained from this processwas more than 97% which was further confirmed by RP-HPLC analysis. Finally, 82 mg of purified rhTα1 was obtained from 1 liter culture. Perhapsthis simple process could provide an effective method for our purpose.The experimental results proved that the design of the molecular structureof the fusion protein was rational. This method overcomed the problem thatsmall molecule peptides expressed in E.coli was difficult. The purificationprocess is simple, rapid and easy to amplify for industrial production. Itprovided a good reference for large expression and purification of rhTα1 withbacteria.