Chinese Journal of Magnetic Resonance ›› 2015, Vol. 32 ›› Issue (1): 1-11.doi: 10.11938/cjmr20150101

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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

  
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).

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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