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背景:瘦素是脂肪组织产生的一种激素,具有广泛的生理作用,也是近年能量代谢研究的热点之一,所用瘦素主要是大肠杆菌的表达产物,后者以不溶解的包含体形式存在,需要通过烦琐的复性过程才能获得具有生物学活性的人瘦素。目的:为获得天然溶解形式的人瘦素,探索其非亲和层析纯化条件,为进一步可能的产业化提供依据。设计:实验观察。单位:一所医学院的生化教研室分子生物学实验室。材料:选择强阴离子交换剂(SepharoseQ)和疏水介质(PhenylSepharose6)在不同条件下进行层析的方法,以尽可能除去样品中的杂质。方法:将培养获得的上清液在pH7.5的条件下首先经过Q柱柱层析纯化,收集目的蛋白;随后在高盐犤mol/L(NH4)2SO4犦状态下通过疏水层析进一步纯化。主要观察指标:经凝胶扫描分析,纯化前的培养上清液人瘦素纯度为42.3%,Q柱柱层析纯化后纯度达到89.6%,疏水层析进一步纯化后纯度达到96.2%。结果:纯化产物在SDS-PAGE上呈现单一条带。经凝胶扫描分析,纯化前的培养上清液人瘦素纯度为42.3%,Q柱柱层析纯化后纯度达到89.6%,疏水层析进一步纯化后纯度达到96.2%。结论:通过强阳离子交换层析和疏水层析的合并应用,可将毕赤酵母表达系统表达的人瘦素进行有效纯化,纯度可达镍柱亲和层析的纯化结果,为进一步可能的产业化生产?
BACKGROUND: Leptin is a hormone produced by adipose tissue and has a wide range of physiological functions. It is also one of the hot topics in energy metabolism research in recent years. Leptin is mainly expressed in Escherichia coli, and the latter exists as an insoluble inclusion body , Need to go through the cumbersome process of refolding to obtain biologically active human leptin. OBJECTIVE: To obtain human leptin in its naturally-soluble form and to explore its purification conditions by non-affinity chromatography, providing the basis for further possible industrialization. Design: experimental observation. Unit: a biomedical laboratory of molecular biology laboratory. Materials: SepharoseQ and PhenylSepharose6 were selected for chromatography under different conditions to remove as much as possible the impurities in the sample. Methods: The supernatant obtained from the culture was first purified by Q column chromatography at pH 7.5 to collect the target protein, and then further purified by hydrophobic chromatography in the condition of high salt 犤 mol / L (NH4) 2SO4 犦. MAIN OUTCOME MEASURES: Human leptin purity was 42.3% after purification by gel scanning analysis. Purity was 89.6% after purification by Q column chromatography and 96.2% after further purification by hydrophobic chromatography. Results: The purified product showed a single band on SDS-PAGE. After gel-scanning analysis, the purity of human leptin before purification was 42.3%, the purity was 89.6% after purification by Q column chromatography, and the purity reached 96.2% after further purification by hydrophobic chromatography. Conclusion: The combination of strong cation exchange chromatography and hydrophobic chromatography can effectively purify human leptin expressed in the expression system of Pichia pastoris and achieve the purity of nickel column affinity chromatography. As a result, Production?