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Mar. Drugs 2015, 13(6), 3388-3406; doi:10.3390/md13063388

Construction of Escherichia coli Mutant with Decreased Endotoxic Activity by Modifying Lipid A Structure

1
Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
3
Department of Bioengineering, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
*
Authors to whom correspondence should be addressed.
Academic Editor: Antonio Molinaro
Received: 31 March 2015 / Revised: 18 May 2015 / Accepted: 19 May 2015 / Published: 27 May 2015
(This article belongs to the Special Issue Marine Lipopolysaccharides)
View Full-Text   |   Download PDF [1349 KB, uploaded 27 May 2015]   |  

Abstract

Escherichia coli BL21 (DE3) and its derivatives are widely used for the production of recombinant proteins, but these purified proteins are always contaminated with lipopolysaccharide (LPS). LPS is recognized by the toll-like receptor 4 and myeloid differentiation factor 2 complex of mammalian immune cells and leads to release of pro-inflammatory cytokines. It is a vital step to remove LPS from the proteins before use for therapeutic purpose. In this study, we constructed BL21 (DE3) ∆msbB28 pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity. The plasmids harboring pagL and/or lpxE were then introduced into this mutant to further modify the LPS. The new strain (S004) carrying plasmid pQK004 (pagL and lpxE) produced mono-phosphoryated tetra-acylated lipid A, which induces markedly less production of tumor necrosis factor-α in the RAW264.7 and IL-12 in the THP1, but still retains ability to produce recombinant proteins. This study provides a strategy to decrease endotoxic activity of recombinant proteins purified from E. coli BL21 backgrounds and a feasible approach to modify lipid A structure for alternative purposes such as mono-phosphoryl lipid A (MPL) as vaccine adjuvants. View Full-Text
Keywords: Escherichia coli BL21 (DE3); lipid A; pagL; lpxE; protein expression Escherichia coli BL21 (DE3); lipid A; pagL; lpxE; protein expression
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Liu, Q.; Li, Y.; Zhao, X.; Yang, X.; Liu, Q.; Kong, Q. Construction of Escherichia coli Mutant with Decreased Endotoxic Activity by Modifying Lipid A Structure. Mar. Drugs 2015, 13, 3388-3406.

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