Gronski P, Badziong W, Habermann P, List W, Müllner H, Neurohr K J, Tripier D, Seiler F R
Research Laboratories of Behringwerke AG, Marburg/Lahn, W.-Germany.
Behring Inst Mitt. 1988 Aug(83):246-9.
Recombinant human GM-CSF has been expressed as a fusion protein in E. coli in the form of inclusion bodies. Using denaturing agents, acid cleavage and sulfitolysis, the biologically inactive GM-CSF protein could be highly purified and additionally renaturated under suitable reoxidizing conditions. The thorough repair of the two disulfide bridges could be confirmed by sequencing fragments obtained by tryptic digestion. Refolding of the molecule has been studied by CD spectrometry and identity by Western blotting and SDS-PAGE analysis. As could be demonstrated, full biological activity (colony-forming assay with fresh human bone marrow cells) was restored during renaturation of the GM-CSF protein. Further proof of biological equivalence of the E. coli-derived protein with a yeast-derived biologically active rh GM-CSF has been published elsewhere.
重组人粒细胞-巨噬细胞集落刺激因子（GM-CSF）已在大肠杆菌中以包涵体形式表达为融合蛋白。通过使用变性剂、酸裂解和亚硫酸解，可高度纯化无生物活性的GM-CSF蛋白，并在合适的再氧化条件下使其复性。通过对胰蛋白酶消化获得的片段进行测序，可以确认两个二硫键的完全修复。通过圆二色光谱法研究了分子的复性，并通过蛋白质印迹法和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析确定了其同一性。如所证明的，GM-CSF蛋白复性过程中恢复了完全的生物活性（用人新鲜骨髓细胞进行集落形成试验）。大肠杆菌来源的蛋白与酵母来源的具有生物活性的重组人GM-CSF在生物学上等效的进一步证据已在其他地方发表。
