绿色荧光蛋白的原核表达、纯化与荧光分析

THE PROKARYOTIC EXPRESSION,PURIFICATION AND FLUORESCENCE ANALYSIS OF A GREEN FLUORESCENT PROTEIN (GFP)

将绿色荧光蛋白(GFP)基因插入到pET30(a)+构建pET30-GFP表达载体,将重组载体导入大肠杆菌BL-21中,经IPTG诱导产生His-GFP融合蛋白,融合蛋白主要存在于可溶性上清中。用镍柱亲合纯化His-GFP,分子量大约为32.4kD,与预期值相符。荧光分析表明,His-GFP与野生型GFP荧光激发及发射波长基本相同,荧光性质稳定,比常用的荧光素(FITC)具有更强的抗荧光淬灭能力。通过此方法制备的绿色荧光蛋白可用于食用色素的开发等。

A pET30a-GFP recombinant plasmid was constructed by inserting GFP gene into pET30a vector, and was transformed into E. Coli. BL-21. After induced by IPTG, the fusion protein （His-GFP） was expressed mainly in the suspension. His-GFP was purified by Ni resins affinity chromatography. The molecular of purified His-GFP was about 32.4 kD as that of deduced from gene. The fluorescence wavelength of His-GFP is similar as that of wildness. The fluorescence characteristic of His-GFP is stable and His-GFP has stronger anti-fluorescence quenching than that of the regular fluorescence reagent （FITC）. The GFP produced by this method could be used as edible pigment.