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

张岩; 赵玢; 杨中正; 申杰; 胡伟; 蓝文贤; 吴厚铭; 曹春阳

doi:10.11938/cjmr20150101

波谱学杂志 >

2015 , Vol. 32 >Issue 1: 1 - 11

DOI: https://doi.org/10.11938/cjmr20150101

研究论文

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

张岩 ,
赵玢 ,
杨中正 ,
申杰 ,
胡伟 ,
蓝文贤 ,
吴厚铭 ,
曹春阳

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State Key Laboratory of Bio-organic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, China

*通讯联系人：曹春阳，电话: 021-54925491; 传真: 021-64166128; E-mail: ccao@mail.sioc.ac.cn.

收稿日期: 2014-02-11

修回日期: 2015-01-08

网络出版日期: 2015-03-05

基金资助

The National Basic Research Program of China (2011CB966300), the National Natural Science Foundation of China (30970595, 21472229 and 20921091).

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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

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

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State Key Laboratory of Bio-organic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, China

CAO Chun-yang, Tel: +86-21-54925491; Fax: 86-21-64166128; E-mail: ccao@mail.sioc.ac.cn. *Corresponding author.

Received date: 2014-02-11

Revised date: 2015-01-08

Online published: 2015-03-05

Supported by

The National Basic Research Program of China (2011CB966300), the National Natural Science Foundation of China (30970595, 21472229 and 20921091).

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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

本文引用格式

张岩 , 赵玢 , 杨中正 , 申杰 , 胡伟 , 蓝文贤 , 吴厚铭 , 曹春阳 . 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[J]. 波谱学杂志, 2015 , 32(1) : 1 -11 . DOI: 10.11938/cjmr20150101

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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