Tightly regulated and high level expression vector construction for Escherichia coli BL21 (DE3)

Amit Kumar Chaudhary; Eun Yeol Lee

Journal of Industrial and Engineering Chemistry, Vol.31, 367-373, November, 2015

DOI10.1016/j.jiec.2015.07.011 Export Citation

Tightly regulated and high level expression vector construction for Escherichia coli BL21 (DE3)

Amit Kumar Chaudhary, and Eun Yeol Lee^†

Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do 446-701, Republic of Korea

E-mail:

A 474-bp ds-DNA linker-A containing all bio-parts was designed to construct tightly inducible expression vector, pNB1, for Escherichia coli. The cymR repressor expressed constitutively under pGapA promoter binds cumate operator, thus repressing the expression of target gene under T7 promoter. The potential of pNB1 was evaluated by comparing the fluorescence of green fluorescence proteins (GFP) in pNB1 and other expression system induced with cumate and isopropyl b-D-1-thiogalactopyranoside (IPTG), respectively. Compared to IPTG, cumate was found to be less toxic to bacterial cells with 2.5-fold increment in GFP expression. In addition, pNB1 was efficient in expressing GFP at low temperature.

Keywords:Cumate;cmt and cym Operons;IPTG;Protein expression;pNB1 vector

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[9] Palomares LA, Estrada-Mondaca S, Ramirez OT, Methods Mol. Biol., 267, 15 (2004)

[10] Figge J, Wright C, Collins CJ, Roberts TM, Livingston DM, Cell, 52, 713 (1988)

[11] Su TZ, Schweizer H, Oxender DL, Gene, 90, 129 (1990)

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[13] Kogure T, Wakisaka N, Takaku H, Takagi M, J. Biotechnol., 129, 502 (2007)

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[16] Baneyx F, Curr. Opin. Biotechnol., 10, 411 (1999)

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