基于谷胱甘肽-磁性微粒的GST融合蛋白纯化体系的建立

Purification of glutathione-S-transferase fusion protein by glutathione coupled magnetic particles

摘要:

本研究将还原型谷胱甘肽(GSH)共价结合在异硫氰酸根末端磁性微粒表面, 制备了具有超顺磁性的谷胱甘肽-磁性微粒亲和介质, 以表面修饰有谷胱甘肽的磁性微粒为载体, 建立了谷胱甘肽巯基转移酶(GST)融合蛋白的纯化体系。对100 mL细胞裂解液纯化体系所需磁性微粒用量、谷胱甘肽-磁性微粒与细胞裂解液的孵育时间、清洗条件等进行了优化。以聚丙烯酰胺凝胶电泳对融合蛋白的纯度进行了检测, Bradford方法对融合蛋白进行了定量测定, 对纯化得到的目的蛋白进行了Western blotting分析。结果表明, 每毫克异硫氰酸根末端磁性微粒对GSH的固定化容量为150 mg, 10 mg谷胱甘肽-磁性微粒可满足100 mL细胞裂解体系中目的蛋白的纯化, 最佳孵育时间为40 min, 对GST融合蛋白的平均纯化量为516 mg。本方法快速、简便, 基于磁性微粒的分离还可实现自动化, 对GST融合蛋白的纯化具有很好的应用前景。

Abstract:

We established a purification system for glutathione-S-transferase (GST) fusion protein using glutathione coupled magnetic particle. Glutathione was coupled covalently to the surface of magnetic particles with isothiocyanate functional groups. Cell lysate, containing the fusion protein, was then incubated with these glutathione coupled magnetic particles at room temperature. Unbound and non-specifically bound proteins were removed by wash steps. Subsequently, the GST-fusion protein was eluted from the magnetic particles by the addition of reduced glutathione. The resulting fusion protein was tested for purity using SDS-PAGE and demonstrated by Western blotting. The concentration of the fusion protein was measured by Bradford method. Both the conditions for incubation and washing were optimized. The results showed that 150 μg glutathione could be bound on 1 mg of particle surface and 10 mg of the glutatione-coupled magnetic particles was suitable for 100 μL lysate, the optimal incubation time for reaction between particles and lysate was 40 min. The magnetic particles could help purify efficiently GST-fusion protein with a yield of around 516 μg fusion protein per 10 mg particles. Magnetic particles can be successfully used in a simple, rapid and reliable method for the purification of GST-fusion proteins.