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目的获得高活性的聚乙二醇定点单修饰的产物PEG-rmhG-CSF-Cys176。方法用聚乙二醇马来酰亚胺基和高活性的重组人粒细胞集落刺激因子突变体(rmhG-CSF)的半胱氨酸(Cys)基团特异性结合,并用离子交换层析分离纯化修饰产物,通过高效凝胶过滤色谱检测纯度,MTT法检测体外的生物活性。结果蛋白纯度为97%,体外生物活性为0.8×108IU·mg-1。结论半胱氨酸反应性PEG和游离Cys的结合是在rmhG-CSF活性区以外的位置C末端,修饰后的蛋白活性基本未受影响。
OBJECTIVE To obtain PEG-rmhG-CSF-Cys176, a highly active polyethylene glycol targeted single modification. Methods Specific binding of polyethylene glycol maleimide groups to the cysteine (Cys) group of a highly active recombinant human granulocyte colony-stimulating factor mutant (rmhG-CSF) and separation by ion exchange chromatography The purified product was purified by high performance gel filtration chromatography and the biological activity was detected by MTT assay. Results The protein purity was 97% and the in vitro biological activity was 0.8 × 10 8 IU · mg -1. Conclusions The binding of cysteine-reactive PEG to free Cys is at the C-terminal position outside the active region of rmhG-CSF, and the modified protein activity is basically unaffected.