融合蛋白表达过程中N末端蛋白对下游蛋白正确折叠的影响

Expression and Molecular Dynamics of Recombinant Ganoderma Lucidum Immunoregulatory Protein-IgG Fc (LZ8-Fc) Fusion Protein in Pichia pastoris

将灵芝免疫调节蛋白(LZ-8)与免疫球蛋白G的Fc片段(IgG Fc)进行融合表达,通过Protein A亲和层析结合Superdex 75分子筛层析获得了高纯度LZ8-Fc.凝血活性实验结果表明,LZ-8与Fc融合表达在一定程度上影响了其免疫活性;利用分子动力学和圆二色光谱等技术研究了LZ8-Fc的蛋白折叠及组装方式,发现LZ8-Fc的组装方式与LZ-8相同,但是N末端的LZ-8可能影响下游IgG Fc片段的正确折叠,表明在融合蛋白制备过程中,位于N末端的蛋白对下游蛋白的正确折叠起到重要作用,这为融合蛋白制备技术的优化及应用过程中的稳定提供了重要的理论依据.

Ganoderma lucidum immunoregulatory protein（LZ-8） was recombinant expressed in Pichia pastoris by prolonging its in vivo half-life through genetic fusion with human IgG-Fc fragment. High purify of LZ8-Fc was obtained by affinity chromatography and size-exclusion chromatography. The result of hemagglutination reaction indicated that LZ-8＇s immunoregulatory activity was inhibited by IgG Fc fragment. Molecular dyna-mics simulation combined with SEC-HPLC and CD proved that LZ8-Fc assembled itself in the same way with LZ-8, dimer in natural state. All of the results suggested that LZ-8 in N-terminal affected IgG-Fc fragment in downstream, which is supposed an important issue in the recombinant expressing of fusion protein. The results will provide a solid basis for preparing and optimizing technology of fusion protein.