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目的 :探索原核基因工程产品质量控制的指标。方法 :以重组粒细胞集落刺激因子(G CSF)为模型 ,将其中构象不均一组分进行分离 ,并对每个成分进行鉴定。结果 :重组人G CSF在大肠杆菌中以包涵体形式表达 ,经纯化和复性后 ,SDS PAGE鉴定为单一蛋白带 ,但在非变性PAGE中出现 3条带 ,继而采用制备型非变性PAGE成功地将 3种成分分离。 3种成分在同样的缓冲体系中分别存放 3个月 ,仍未见互变异构现象。经激光解析飞行时间质谱检测 ,3种成分相对分子质量相同 ,测序表明 ,N端连续的 5个氨基酸残基排列完全相同。结论 :3种成分为重组蛋白的不均一折叠形成的构象异构体。
Objective: To explore the quality control of prokaryotic genetic engineering products. Methods: G CSF was used as a model to separate the inhomogeneous conformations and identify each component. Results: Recombinant human G CSF was expressed as a inclusion body in E. coli. After purification and refolding, the recombinant protein was identified as a single protein band by SDS PAGE. However, 3 bands appeared in the native PAGE, followed by the preparation of non-native PAGE The three components will be separated. Three components in the same buffer system were stored for 3 months, still no tautomerism. By laser desorption / ionization time of flight mass spectrometry, the relative molecular masses of the three components were the same. Sequencing showed that the five consecutive N-terminal amino acid residues were arranged in exactly the same order. CONCLUSION: The three components are the conformational isomers formed by inhomogeneous folding of recombinant proteins.