波谱学杂志 ›› 2015, Vol. 32 ›› Issue (1): 1-11.doi: 10.11938/cjmr20150101

• 研究论文 • 上一篇    下一篇

原核表达过程中非目标蛋白质识别与确认的方法:NRSF/REST蛋白功能结构域ZnF2-8原核表达过程中b-内酰胺酶的确认

ZHANG Yan,ZHAO Bin,YANG Zhong-zheng,SHEN Jie,HU Wei,LAN Wen-xian,WU Hou-ming,CAO Chun-yang*   

  1. State Key Laboratory of Bio-organic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, China
  • 收稿日期:2014-02-11 修回日期:2015-01-08 出版日期:2015-03-05 发布日期:2015-03-05
  • 作者简介:*通讯联系人:曹春阳,电话: 021-54925491; 传真: 021-64166128; E-mail: ccao@mail.sioc.ac.cn.
  • 基金资助:
    The National Basic Research Program of China (2011CB966300), the National Natural Science Foundation of China (30970595, 21472229 and 20921091).

Methods for Identification and Characterization of Protein Unexpectedly Expressed in Escherichia Coli: A Case Study Involving b-Lactamase Observed during the Expression of Zinc Finger 2-8 of NRSF/REST

  
中国科学院 上海有机化学研究所,生命有机国家重点实验室,上海 200032
  

  1. State Key Laboratory of Bio-organic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, China
  • Received:2014-02-11 Revised:2015-01-08 Online:2015-03-05 Published:2015-03-05
  • About author:CAO Chun-yang, Tel: +86-21-54925491; Fax: 86-21-64166128; E-mail: ccao@mail.sioc.ac.cn. *Corresponding author.
  • Supported by:
    The National Basic Research Program of China (2011CB966300), the National Natural Science Foundation of China (30970595, 21472229 and 20921091).

摘要:

大肠杆菌常被用来大量快速制备重组蛋白质.但是,在原核表达目标蛋白质时非目标蛋白质经常会意外表达.有时这些非目标蛋白质也非常有使用价值,但是最终确认这些非目标蛋白质的过程昂贵又及其耗时.基于此,该文发展了一个新的基于二维核磁共振波谱技术、X-单晶衍射技术、结合其他生物化学方法,确认在原核表达神经限制性沉默因子NRSF/REST蛋白(该蛋白能够特异性识别神经限制性沉默因子RE1 dsDNA及神经限制性激活因子dsRNA,以调节神经元干细胞的发育)功能结构域ZnF2-8时非目标蛋白b-内酰胺酶(b-lactamase).

关键词: NRSF/REST, ZnF2-8, 核磁共振(NMR), b-内酰胺酶, 意外表达

Abstract:

Escherichia coli (E. coli) is often used to produce recombinant proteins rapidly with high yield. However, the bacteria expresses non-target proteins unexpectedly in many cases, some of which may later be proven useful. Full characterization of these unpredicted proteins are usually expensive and time-consuming. In this study, we used E. coli to express neuron- restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) functional motif ZnF2-8, which is involved in the interaction of NRSF/REST with neuron-restrictive silencer element (NRSE/RE1) dsDNA or small non-coding dsRNA for neuron gene transcriptional repression or activation. Overexpression of a non-target protein was observed. Two-dimensional 1H-15N HSQC NMR spectroscopy, X-ray crystallography and other biochemical assays were used in combination to characterize the non-target protein to be b-lactamase.

Key words: NRSF/REST, ZnF2-8, NMR spectroscopy, b-lactamase, unexpectedly overexpress

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