Chinese Journal of Magnetic Resonance >

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

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

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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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

Cite this article

ZHANG Yan , ZHAO Bin , YANG Zhong-zheng , SHEN Jie , HU Wei , LAN Wen-xian , WU Hou-ming , CAO Chun-yang . 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[J]. Chinese Journal of Magnetic Resonance, 2015 , 32(1) : 1 -11 . DOI: 10.11938/cjmr20150101

References

[1]  Berrow N S, Alderton D, Sainsbury S, et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications[J]. Nucleic Acids Res, 2007, 35: e45.


 


[2]  Cabrita L D, Dai W W, Bottomley S P. A family of E-coli expression vectors for laboratory scale and high throughput soluble protein production[J]. BMC Biotechnol, 2006, 6: 12.



[3]  Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability[J]. PLoS ONE, 2008, 3: e3647.


 


[4]  Gileadi N A, Burgess-Brown S M, Colebrook G, et al. High Throughput Production of Recombinant Human Proteins for Crystallography//Kobe B, Guss M, Huber T, Eds. Structural Proteomics: High Throughput Methods[M]. New York: Humana Press, 2008: 221－246.



[5]  Graslund S, Nordlund P, Weigelt J, et al. Protein production and purification[J]. Nat Methods, 2008, 5: 135－146.


 


[6]  Scheich C, Kummel D, Soumailakakis D, et al. Vectors for co-expression of an unrestricted number of proteins[J]. Nucleic Acids Res, 2007, 35: e43.



[7]  Cormier C Y, Mohr S E, Zuo D, et al. Protein structure initiative material repository: an open shared public resource of structural genomics plasmids for the biological community[J]. Nucleic Acids Res, 2010, 38: D743－D749.


 


[8]  Blommel P G, Martin P A, Wrobel R L, et al. High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system[J]. Protein Expr Purif, 2006, 47: 562－570.



[9]  Li M Z, Elledge S J. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC[J]. Nat Methods, 2007, 4: 251－256.


 


[10]  Walhout A J M, Temple G F, Brasch M A, et al. GATEWAY recombinational cloning: Application to the cloning of large numbers of open reading frames or ORFeomes[J]. Methods Enzymol, 2000, 328: 575－592.



[11]  Stols L, Gu M Y, Dieckman L, et al. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site[J]. Protein Expr Purif, 2002, 25: 8－15.


 


[12]  Chanda P K, Edris W A, Kennedy J D. A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli[J]. Protein Expr. Purif, 2006, 47: 217－224.



[13]  Ballas N, Mandel G. The many faces of REST oversee epigenetic programming of neuronal genes[J]. Curr Opin Neurobiol, 2005, 15: 500－506.


 


[14]  Eddy S R. Non-coding RNA genes and the modern RNA world[J]. Nat Rev Genet, 2001, 2: 919－929.



[15]  Fire S, Xu M K, Montgomery S A, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391: 806－811.

 


 


 



 


[17]  Kuwabara T, Hsieh J, Nakashima K, et al. A small modulatory dsRNA specifies the fate of adult neural stem cells[J]. Cell, 2004, 116: 779－793.





 


[19]  Chowdhury S K, Katta V, Chait B T. Electrospray ionization mass spectrometric peptide mapping: A rapid, sensitive technique for protein structure analysis[J]. Biochem Bioph Res Commun, 1990, 167(2): 686－692.



[20]  Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode[J]. Method Enzymol, 1997, 276: 307－326.


[21]  Emsley P, Cowtan K. Developments in the CCP4 molecular-graphics project[J]. Acta Crystallogr D Biol Crystallogr, 2004, 60: 2 126－2 132.


 


[22]  Afonine P V, Mustyakimov M, Grosse-Kunstleve R W, et al. Joint X-ray and neutron refinement with phenix.refine[J]. Acta Crystallogr D Biol Crystallogr, 2010, 66: 213－221.



[23]  Abraham E P, Chain E B. An enzyme from bacterial able to destroy penicillin[J]. Nature, 1940, 146: 837－837.


 


[24]  Frere J M. β-lactamase and bacterial resistance to antibiotics[J]. Molec Microbiol, 1995, 16: 385－395.


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