上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

重组大鼠α2巨球蛋白的表达、纯化及结晶

冯玲玲，周爱武   

  1. 上海交通大学 医学院细胞分化与凋亡教育部重点实验室，上海 200025
  • 出版日期:2016-12-28 发布日期:2016-12-29
  • 通讯作者: 周爱武，电子信箱：aiwu.zhou@googlemail.com。
  • 作者简介:冯玲玲（1983—），女，助理实验师，硕士；电子信箱：llfeng@shsmu.edu.cn。
  • 基金资助:

    国家自然科学基金（31170724）

Expression, purification, and crystallization of recombinant rat α2 macroglobulin

FENG Ling-ling, ZHOU Ai-wu   

  1. Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
  • Online:2016-12-28 Published:2016-12-29
  • Supported by:

    National Natural Science Foundation of China,31170724

PDF (PC)

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摘要：

目的 ·获取高纯度的大鼠α2巨球蛋白(Rα2M)，并对蛋白结晶进行筛选，以期获取高分辨率衍射数据，为后续结构生物学研究奠定基础。方法  ·将Rα2M基因克隆到真核表达载体pCEP4中，使用PEI介导的瞬时转染方法在HEK293EBNA细胞中表达重组蛋白Rα2M。采用阴离子柱交换层析和镍柱亲和层析技术从表达培养基中提纯Rα2M，对纯化蛋白进行结晶筛选和优化。结果 ·经纯化后，得到相对分子质量约为180 000的重组蛋白Rα2M的全长蛋白，以及被切割后的大片段和小片段。通过结晶筛选和优化后，得到了Rα2M的蛋白晶体。结论 ·获得了可用于晶体衍射的Rα2M蛋白晶体，为后续的结构解析奠定了基础。

关键词: &alpha, 2巨球蛋白；真核表达；纯化；结晶

Abstract:

Objective · To obtain high purity rat α2 macroglobulin (Rα2M) and screen the protein crystals in order to collect high-resolution diffraction data for subsequent structural biology research. Methods · The gene for encoding Rα2M was cloned into the eukaryotic expression vector pCEP4. Recombinant Rα2M was expressed in HEK293EBNA cells using PEI-mediated transient transfection method. Rα2M was purified with anion exchange column and nickel affinity chromatography and purified Rα2M was crystallized, screened, and optimized. Results · The recombinant full length Rα2M protein with a relative molecular mass of about 180 000 was obtained after purification, as well as small and large fragments after slicing. Rα2M protein crystals were obtained after crystallization screening and optimization. Conclusion · The Rα2M crystals that can be used for crystal diffraction were obtained. This work lays a foundation for subsequent elucidation of the crystal structure of Rα2m.

Key words:  α2 macroglobulin, eukaryotic expression, purification, crystallization