重组人糜蛋白酶表达、纯化和性质研究

摘要

目的：研究重组人糜蛋白酶（human Chymotrypsin）在大肠杆菌中的表达、纯化和部分性质。方法大肠杆菌BL 21（DE 3）高效表达目的蛋白，表达形式为包涵体，复性后的重组蛋白，通过阳离子交换层析纯化，对纯化蛋白进行性质研究。结果 SDS-PAGE分析重组蛋白酶原大小约为30 KD， CM-FF阳离子交换层析纯化后最终活性回收率为93.7%，重组酶在pH 3～pH5范围内稳定，温度稳定性好，以N-苯甲酰-L-酪氨酸乙酯(n-benzoyl-l-tyrosine ethyl ester,BTEE)作为底物时的Km值为0.067 mmol/L，紫外最大吸收波长为281 nm。结论重组人糜蛋白酶工程菌成功表达目的蛋白，并通过复性、激活后获得了活性蛋白，建立了生产该酶的方法。%Objective To study the prokaryotic expression, puriifcation and properties of recombinant human Chymotrypsin. Methods The protein was highly expressed in E.coliBL 21 (DE 3) as inclusion body. After refolding and activated with trypsin, the activated protein was obtained and purified with ion-exchange chromatography(CM-FF), some properties of the recombinant human chymotrypsin was investigated. Results The molecular weight of chymotrypsinogen was about 30 KD in SDS-PAGE, total activity recovery rate of CM-FF puriifcation was 93.7%. The recombinant chymotrypsin kept stable from pH 3 to pH 5, and owned good temperature stability. Km was 0.067 mmol/L with BTEE as a substrate. The UV maximum absorption wavelength was 281 nm.Conculsion The recombinant human enzyme was expressed successfully, and a feasible production method to get a high activity of the recombinant human chymotrypsin was established.