交叉酶切联合MALDI-TOF/MS分析重组蛋白二硫键分布的方法建立

摘要

以Kringle环为分子模型,应用MALDI-TOF/MS联合特异性蛋白酶酶切技术,探讨建立蛋白质二硫键分布和定位分析的研究方法.采用pET22b(+)原核表达体系诱导表达重组人纤溶酶原K5蛋白(rhK5),经Ni2+ - NTA resin亲和层析纯化,并通过SDS-PAGE和Western-blot进行初步鉴定；采用MTT法分析rhK5对微血管内皮细胞的抑制活性；联合应用交叉酶切(Trypsin Gold单切或Trypsin Gold及Endoproteinase ASP-N双酶切)和基质辅助激光解析-飞行时间质谱(MALDI-TOF/MS)分析rhK5的二硫键分布；并应用生物信息学方法和圆二色谱法进一步验证rhK5二级结构.应用原核体系高效表达具有生物活性的rhK5蛋白,经交叉酶切联合MAL-DI-TOF/MS证实rhK5中的6个半胱氨酸正确配对形成了3对二硫键(Cys462:541,Cys483:524和Cys512:536),圆二色谱法测定发现rhK5为典型的β折叠型结构.上述研究结果为分析基因工程重组蛋白质中半胱氨酸配对及二硫键分布提供了方法学基础.%In order to establish a method for analyzing the distribution and localization of disulfide in re-combinant protein by specific protease digestion combined with MALDI-TOF/MS. The pET22b ( + ) pro-karyotic system was harnessed to generate recombinant protein of human plasminogen kingle ( rhK5 ) which was purified by Ni2+ - NTA resin affinity chromatography. SDS - PAGE and Western blot analysis were used to identify the recombinant protein. The disulfide bridging conformation and dimensional structure of rhK5 were predicted by 3D-JIGS AW Comparative Modeling Server (UK) and the disulfide bond distribution of rhK5 was confirmed by orthogonal enzymatic digestion and MALDI-TOF/MS. The seconda-ry structure of rhK5 was analyzed by circular dichroism. Enzymatic digestion and MALDI-TOF/MS analysis demonstrated the presence of disulfide bridges among Cys462-Cys541, Cys483-Cys524 and Cys512-Cys536 in rhK5. Circular dichroism showed that rhK5 adopted the shape characteristic of a β - sheet. Our studies provide methodological basis for analyzing the pairing and distribution of disulfide bond of genetic engineering recombinant proteins.