高等学校化学学报

高等学校化学学报 ›› 2003, Vol. 24 ›› Issue (3): 386.

• 论文 • 上一篇    下一篇

具有生物活性的人带3蛋白胞质片段融合蛋白的克隆、表达与纯化

郑玉娟, 邢菲菲, 孙立伟, 刘立岩, 赵大庆, 倪嘉缵   

  1. 中国科学院长春应用化学研究所, 稀土化学与物理国家重点实验室, 长春 130022
  • 收稿日期:2002-04-16 出版日期:2003-03-24 发布日期:2003-03-24
  • 通讯作者: 赵大庆(1963年出生),男,博士,研究员,博士生导师,从事生物无机化学及分子生物学研究.
  • 基金资助:

    国家自然科学基金重大项目(批准号:29890280)资助

Cloning, Expression and Purification of a Functional Fusion Protein with Cytoplasmic Domain of Human Band 3

ZHENG Yu-Juan, XING Fei-Fei, SUN Li-Wei, LIU Li-Yan, ZHAO Da-Qing, NI Jia-Zuan   

  1. Key Laboratory of Rare Earth Chemistry and Physics, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
  • Received:2002-04-16 Online:2003-03-24 Published:2003-03-24

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被引次数

3

摘要： 用PCR法从质粒pHB3中扩增了人红细胞带3蛋白胞质片段(CDB3)基因.PCR产物经限制性内切酶切割后与多克隆位点处带有编码6个组氨酸序列的高效表达载体pET28b连接,构建为重组子pCDBHistag.重组子经酶切及序列测定后在大肠杆菌BL21(DE3)中获得高效表达,可溶性目的蛋白占菌体总蛋白的40%左右.C端带有6个连续组氨酸的带3蛋白胞质片段作为融合蛋白不仅可以降低宿主菌蛋白酶对其水解程度,而且简化了目的蛋白的纯化过程.经一步螯合Ni2+的亲和层析获得了电泳纯的带3蛋白胞质片段融合蛋白.活性测定结果表明,带3蛋白胞质片段融合蛋白能够抑制醛缩酶(Aldolase)活性的70%,与文献报道的人红细胞内带3蛋白胞质片段具有相同的功能.

关键词: 带3蛋白胞质片段, 融合蛋白, 亲和层析

Abstract: The cytoplasmic domain of human erythrocyte band 3(CDB3) gene was amplified by standard PCR method from plasmid pHB3 and constructed to be a recombinant plasmid pCDBHistag with an expression vector pET28b. Recombinant CDB3 fusion protein with Polyhistidine-tag was strongly expressed as soluble protein in E coli BL21(DE3) after induction with isopropyl-β-D-thiogalactopyranoside. Affinity chromatograpy chelating with Ni 2+ was employed to purify the recombinant fusion CDB3 protein. About 70% of aldolase activity could be inhibited by purified recombinant CDB3 similar as the result in human erythrocyte. It suggested that aldolase could bind with the purified CDB3 fusion protein in vitro. The high-level expression and easy purification of this recombinant protein should permit thorough studies of the interaction mechanism of CDB3 with its peripheral binding proteins.

Key words: Cytoplasmic domain of band 3, Fusion protein, Affinity chromatography

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