Chinese Medical Sciences Journal ›› 2018, Vol. 33 ›› Issue (1): 1-8.doi: 10.24920/11805

• 论著 •    下一篇

鼠源Nectin-like 4糖蛋白在293ET细胞系中的重组表达与纯化


  1. 中国医学科学院基础医学研究所,北京协和医学院基础学院,国家医学分子生物重点实验室,北京 100005
  • 收稿日期:2017-10-23 出版日期:2017-03-17 发布日期:2018-03-07

Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line

Li Dongdong,An Tai,Liu Xiao,Yin Bin,Peng Xiaozhong(),Shu Pengcheng()   

  1. State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China
  • Received:2017-10-23 Published:2017-03-17 Online:2018-03-07
  • About author:Corresponding author Tel: 86-10-69156434, Fax: 86-10-65240529; E-mail: Xiaozhong Peng|Corresponding author Tel: 86-10-69156434, Fax: 86-10-65240529; E-mail: Pengcheng Shu

摘要: 目的 筛选可以高水平表达Nectin-like 4 蛋白的稳定细胞系。方法 首先,将Necls胞外区的cDNA序列插入到修饰载体pAPtag5上碱性磷酸酶(AP)的N端,使其融合表达。其次,在293ET细胞中分泌表达Necls-AP融合蛋白,并通过AP活性和蛋白质免疫印迹等方法检测蛋白表达情况。然后,通过在细胞培养过程中加入N-糖链形成抑制剂以抑制复杂糖链的形成。在纯化的蛋白溶液中加入糖苷酶去除剩余的糖链。最后,使用人鼻病毒3C蛋白酶将融合蛋白上的AP蛋白切除,通过凝胶排阻色谱将该蛋白与目的蛋白分离。最终获得的目的蛋白在280nm波长下检测获得其浓度,通过SDS-PAGE电泳结果检测分析蛋白纯度。结果 通过利用重组载体上AP蛋白标签的颜色反应,我们筛选得到高水平表达Necl-4蛋白的稳转细胞系。在对目的蛋白进行了去糖基化等处理,我们最终可在1L的细胞培养上清中纯化获得4mg可溶、有活性的Necl-4蛋白,且蛋白纯度在95%左右。结论 使用AP蛋白—哺乳动物细胞表达系统,通过对AP蛋白的活性分析,我们快速筛选得到蛋白高表达量的稳转细胞系。在细胞培养中添加糖链抑制剂以及用糖苷酶处理纯化后的蛋白,可以得到了毫克级的去糖基化蛋白Necl-4。.本文中所使用的方法,对于表达、纯化得到糖蛋白用于进一步的结构和功能研究提供了思路。

关键词: Nectin-like 免疫球蛋白, 膜蛋白, 快速筛选法, 糖基化, 哺乳动物细胞

Abstract: Objective To screen the transient and stable cell lines with high production of Nectin-like 4 (Necl-4) protein.Methods First, cDNA sequences encoding the extracellular domain of Necls were cloned into the modified vector pAPtag at the N terminus of alkaline phosphatase (AP) for fusion expression. Next, 293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis. Then, by adding N-glycosylation processing inhibitor kifunensine into the medium, complex glycan was inhibited to generate. The residual glycan of purified protein was removed by endoglycosidase H. Finally, AP protein was removed by using human rhinovirus protease and size exclusion chromatography. The concentration of purified Necl-4 protein was monitored by measuring the absorbance at 280 nm and analyzed by SDS-PAGE.Results The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector. The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%.Conclusions By using modified AP mammalian protein expression system, we can easily screen the high productive stable cell lines by using AP activity assay. By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H, we can obtain deglycosylated Necl-4 protein in milligram quantities. Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.

Key words: Nectin-like immunoglobulin, membrane protein, fast and easy screening, glycosylation, mammalian cell

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