Shake Flask Expression Of Recombinant Human Tumor Necrosis Factor (of Rhtnf-alpha) In Escherichia Coli, Refolding And Purification,

In recent years, protein folding liquid chromatography (PFLC) has become essential means of renaturation and purification of recombinant protein drugs. It can significantly improve the renaturation efficiency of the target proteins and the denatured proteins can be renatured with simultaneous purified. In this paper, the fermentation conditions for rhTNF-αexpressed by E.coli were studied. The highly expressing and highly outputing rhTNF-αcan be obtained under the optimal fermentation conditions. In addition, the renaturation with simultaneous purification of rhTNF-αwas investigated with high performance hydrophobic interaction chromatography ( HPHIC ) and high performance ion exchange chromatography ( HPIEC ) respectively.Protein folding and the new methods to renature the denatured proteins developed recently were reviewed firstly. The fermentation, renaturation and purification of rhTNF-αand its biological function and clinical application were also introduced. Sepcially, the rentuation and purification of rhTNF-αwas summaried in detail. It contains 91 references.Then the fermentation conditions of rhTNF-αexpressed in E. coli. with shaking-flask were optimized. The effects of culture media, culture temperature, time of inducing, expression times, inoculum volume and composition of culture media on the growth of E.coli were investigated by single factor method and Orthogonal Design in detail based on shaking-flask. The results showed that at the optimum conditions 39.4% rhTNF-αcan be obtained.HPHIC was applied to the renaturation with simultaneous purification of rhTNF-αexpressed by E.coli. The effects of the extract solution, stationary phase, pH value, urea concentration and different ratios of GSH/GSSG in the mobile phase on the renaturation and simultaneous purification of rhTNF-αwith HIC were investigated in detail repectively. The results showed that at the optimum conditions the mass recovery and purity can be obtained to be 38.7 % and 90% repectively, and the specific activity reached to 2.64×10~7.Weak anion exchange chromatography ( WAX ) was applied to the renaturation with simultaneous purification of rhTNF-αexpressed by E.coli successfully. The effects of several factors, including stationary phase, pH value, urea concentration and ratios of GSH/GSSG in the mobile phase on the renaturation and simultaneous purification of rhTNF-αwith WAX were investigated respectively. The results show that at the optimum conditions the mass recovery and purity can be obtained to be 77 % and 95.2% repectively, and the specific activity reached to 2.18×10~8 IU/mg.