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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2011, Vol. 5 Issue (1) : 72-76    https://doi.org/10.1007/s11703-011-1060-z
RESEARCH ARTICLE
Construction of the expression vector and location analysis of thermotolerant endoglucanase in E. coli
Runfang GUO, Kexue GAO, Hongwei YU, Yingmin JIA()
College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, China
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Abstract

To obtain the secreting expression vector, the signal peptide sequence and mature peptide sequence of endoglucanase from BoldItalic KX6 were cloned into the pET28a plasmid. The recombinant vector pET28a/KX6 was transformed BoldItalic Rosetta (DE3), and the transformant was induced by IPTG. The expression products were primarily distributed in the medium fluid of host cell in a soluble form and the activity was higher than that of other fractions. Both location analysis of targeting protein and activity analysis showed that the signal peptide of endoglucanase from BoldItalic KX6 had played a very important role in the secret expression and activity of foreign proteins in the BoldItalic host cell.

Keywords thermotolerant endoglucanase      expression vector      signal peptide      location analysis     
Corresponding Author(s): JIA Yingmin,Email:ymjia@mail.hebau.edu.cn   
Issue Date: 05 March 2011
 Cite this article:   
Kexue GAO,Hongwei YU,Yingmin JIA, et al. Construction of the expression vector and location analysis of thermotolerant endoglucanase in E. coli[J]. Front Agric Chin, 2011, 5(1): 72-76.
 URL:  
https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1060-z
https://academic.hep.com.cn/fag/EN/Y2011/V5/I1/72
Fig.1  Agarose gel electrophoresis of PCR product
Note: M is the marker DL 2000 and 1 is PCR product.
Fig.2  Identification of recombinant plasmid digested by RI/ I
Note: M is the marker DL15000, 1 is recombinant plasmid pET-28a/KX6, and M is the marker DL2000.
Fig.3  SDS-PAGE analysis of different induced time of pET-28a/KX6
Note: M represents the protein marker, 1 represents pET-28a vector, 2 represents pET-28a vector induced by IPTG for 6 h, and 3 to 9 represent recombinant vector pET-28a/KX6 induced by IPTG for 0 to 6 h, respectively.
Fig.4  Localization analysis of recombinant plasmid pET-28a/KX6-expressing protein
Note: M represents the protein marker. Lanes 1 to 5 represent TCP fraction, insoluble cytoplasmic fraction, soluble cytoplasmic fraction, periplasmic fraction, and medium fraction, respectively.
location of target proteinsendoglucanase activity/Utotal activity/%
insoluble cytoplasmic fraction3.780.57
soluble cytoplasmic fraction127.4419.15
periplasmic fraction00
medium fraction534.2480.28
Tab.1  Endoglucanase activity of different fractions
Fig.5  Cogon red–CMCNa plate determination
Note: 1 to 4 represent medium fraction, periplasmic fraction, soluble cytoplasmic fraction, and insoluble cytoplasmic fraction, respectively. 5 is blank and 6 is control.
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