重组融合蛋白Trx-IFN-CSP复性工艺研究

Renaturation Technology of Recombinant Fusion Protein Trx-IFN-CSP

旨在建立并优化融合蛋白Trx-IFN-CSP的复性工艺。对重组融合Trx-IFN-CSP进行体外复性研究,考查p H、温度、蛋白浓度、氧化还原体系及辅助复性小分子等复性条件对融合蛋白重折叠的影响。结果显示,适合Trx-IFN-CSP复性的方法为,反复冻融联合超声破菌获得包涵体;用含有1%Triton X-100、2 mol/L尿素、2%DOC洗涤液初步纯化包涵体;再用6 mol/L盐酸胍溶解液变性包涵体;脉冲加样稀释变性液后4℃条件下梯度透析复性,使用L-Arg辅助复性。经肠激酶切去Trx标签后,每升发酵液最终获得110-130 mg肝靶向干扰素,每批蛋白纯度都在95%以上,比活性在1.9-2.4×108 U/mg之间,制备工艺稳定。

This work is to establish and optimize the method of refolding fusion protein Trx-IFN-CSP. The refolding of recombinant protein in vitro was studied,i.e.,investigating the effects of pH,temperature,protein concentration,redox systems and auxiliary refolding molecules on the refolding process of fusion protein. The results were as below. The proper measure to refold the Trx-IFN-CSP was to obtain the inclusion bodies by repeated combined freeze-thaw with ultrasonic to crack bacteria. The inclusion bodies were preliminarily purified using scrub solution of 1% TritonX-100,2 mol/L urea,and 2% DOC,and then denatured in 6 mol/L guanidine hydrochloride. After the pulse dilution, the renaturation was conducted under 4℃ by gradient dialysis with the assistance of L-Arg. After the Trx-tag was removed by recombinant enterokinase digestion,approximately 110-130 mg of the pure recombinant liver-targeted interferon was obtained from 1 L Escherichia coli culture. Each batch of protein had a purity of over 95% and antibacterial activities were about 1.9-2.4×108 U/mg,thus the technology of preparation was stable.