Recombinant Fusion Protein Of Human IFNα_2a And Thyα1 Expressed In E.coli

Both human interferon α2a (IFNα2a) and thymosin α1 (THYα1) are very useful drugs for treatment of human cancer and some virus infection diseases as well as improving the immunity. Due to the fused IFNα2a and THYα1 is more powerful than the individual of both IFNα2a and THYal medicines during treating human diseases, we considered to construct a fusion protein molecule containing both IFNα2a and THYal by DNA recombination. First of all, based on the three dimension structure of both molecules, a linker with 12 amino acid length was designed to connect to IFNα2a C terminal and THYal N terminal. After that, an expressed vector pCJlOl containing the fusion gene was obtained and transformed into host cell E.coli DH5a.In order to prepare recombinant fusion protein of IFNα2a-THYα1 with the transgenic E. coli, the optimal process for the target protein expression and purification was studied in more detail. The optimum experimentconditions for the genetic engineered E.coli were as following: culture at 37 ℃ for 14 hours and the medium contained yeast abstract 13g /L, peptone 12g/L, Glucose 3g /L, NaCl 1.5g/L, NH4Cl 0.8g/L, MgSO4 0.9g/L, CaCl2 12 mg/L, resulted from the orthogonal design and even design. Based on the above, the inclusion body weight was 2.1 times more than that by usual 2xYT medium with the same engineered stain and protein separation procedures.The heterognous protein expressed in E.coli was usually accumulated as inclusion bodies in the bacterium. Therefore, how to refold correctly proteins at high concentration with high yield is one of critical techniques for recombinant proteins production. Protein refolding by dilution is the most simply method for protein renaturation than others. In this account, the crude inclusin bodies were washed with 2mol/L urea before refolding in order to remove impurities. Then the inclusion bodies redissolved by 7mol/L guanidine hydrochloride contained 10mmol/L DTT. After that, the denatured liquid was added to the renaturation solution by a pulse way at 0℃. After dialysis, the renaturation sample was loaded on DEAE-sepharose columm. The puporsed protein band on SDS-PAGE possessed about 23kD in molecular weight. The biofunction test of the fusion protein showed IFNα2a activity reached 1.1 × 106IU/ml and THYal reached 20%(E-rosette formation rate), respectively. As a result, the bioactivity of the IFNα2a and THYα1 did not interfere with each other. So...