Construction of the expression vector and location analysis of thermotolerant endoglucanase in E. coli

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

References

[1] Huo Y M, He Q W, Zhao S Y, Xu Y F (2008). Prokaryotic expression of OC- I D86 (Oryzacystatin –I D86) gene and analysis of its activity. Chin J Biotechnol, 24(7): 1194–1198 (in Chinese)

[2] Ikemura H, Takagi H, Inouye M (1987). Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. J Biol Chem, 262(16): 7859–7864

[3] Kim S, Lee S B (2008). Soluble expression of archaeal proteins in Escherichia coli by using fusion-partners. Protein Expr Purif, 62(1): 116–119

[4] Lei R Y, Qiao Y H, Yan J D, Yang S, Zhu T H (2008). Soluble expression of recombinant human BMP6 in Escherichia coli and its purification and bioassay in vitro. Chin J Biotechnol, 24(3): 452–459 (in Chinese)

[5] Miller G L (1959). Use of dinitrosalicyclic acid regent for determination of reducing sugar. Anal Chem, 31(3): 426–428

[6] Qiao Y, Mao A J, He Y Z, Liu W F, Dong Z Y (2004). Secreted expression of Trichoderma reesei endo-glucanase II gene in Pichia pastoris and analysis of enzymatic properties. Mycosystema, 23(3): 388–396

[7] Sørensen H P, Mortensen K K (2005). Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact, 4(1): 1–8

[8] Stader J A, Silhavy T J (1990). Engineering Escherichia coli to secrete heterologous gene products. Methods Enzymol, 185: 166–187

[9] Solingen P, Meijer D, Kleij W A, Barnett C, Bolle R, Power S D, Jones B E (2001). Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake. Extremophiles, 5(5): 333–341

[10] Weickert M J, Doherty D H, Best E A, Olins P O (1996). Optimization of heterologous protein production in Escherichia coli. Curr Opin Biotechnol, 7(5): 494–499

[11] Wulff N A, Carrer H, Pascholati S F (2003). Cloning and expression of cellulase Xf818 from Xylella fastidiosa in Escherichia coli. Scientia Agricola (Piracicaba, Braz.), 60 (4): 715–721

[12] Yamamoto T, Okawa N, Endo T, Kaji A (1991). Expression of chimeric ras protein with OmpF signal peptide in Escherichia coli: localization of OmpF fusion protein in the inner membrane. Appl Microbiol Biotechnol, 35(5): 615–621

Acknowledgements

This study was supported by the project of Sic-Tech Plan of Hebei Province, China (No. 07225553) and the Key Science and Technology Project of the 11th Five-Year-Plan of Hebei Province, China (No. 06220106D).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg