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

Liu Q, Li Y, Zhao X, Yang X, Liu Q, Kong Q - Mar Drugs (2015)

Bottom Line: In this study, we constructed BL21 (DE3) ∆msbB28 ∆pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity.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 Article: PubMed Central - PubMed

Affiliation: Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China. p19890528@126.com.

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.

No MeSH data available.


Related in: MedlinePlus

Construction of BL21 (DE3) mutant strain. (A) Map of deletion mutant resulting from knockout of msbB gene; (B) Map of deletion mutant resulting in knockout of pagP gene; (C) The identification result of ∆msbB28 mutant, the result shows that msbB gene was deleted; (D) The identification result of ∆pagP38 mutant, the result shows that pagP gene was deleted.
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marinedrugs-13-03388-f001: Construction of BL21 (DE3) mutant strain. (A) Map of deletion mutant resulting from knockout of msbB gene; (B) Map of deletion mutant resulting in knockout of pagP gene; (C) The identification result of ∆msbB28 mutant, the result shows that msbB gene was deleted; (D) The identification result of ∆pagP38 mutant, the result shows that pagP gene was deleted.

Mentions: Two suicide plasmids pQK001 and pQK002 were constructed to facilitate deletion of msbB and pagP gene in the BL21 (DE3), respectively, and the gene deletions were performed by homologous recombination via SacB-based counter-selection in allelic-exchange experiments [37]. Figure 1 illustrates the chromosomal structures of ∆msbB28 and ∆pagP38 in BL21 (DE3), and their PCR product size when amplified by flanking primers. The intermediate single ∆msbB28 or ∆pagP38 mutants were further confirmed by DNA sequencing. ∆pagP38 was introduced into intermediate mutant ∆msbB28 to yield the mutant S001 (∆msbB28 ∆pagP38), which was hypothesized to result in production of penta-acylated lipid A in this progeny mutant strain (Table 2).


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

Liu Q, Li Y, Zhao X, Yang X, Liu Q, Kong Q - Mar Drugs (2015)

Construction of BL21 (DE3) mutant strain. (A) Map of deletion mutant resulting from knockout of msbB gene; (B) Map of deletion mutant resulting in knockout of pagP gene; (C) The identification result of ∆msbB28 mutant, the result shows that msbB gene was deleted; (D) The identification result of ∆pagP38 mutant, the result shows that pagP gene was deleted.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4483635&req=5

marinedrugs-13-03388-f001: Construction of BL21 (DE3) mutant strain. (A) Map of deletion mutant resulting from knockout of msbB gene; (B) Map of deletion mutant resulting in knockout of pagP gene; (C) The identification result of ∆msbB28 mutant, the result shows that msbB gene was deleted; (D) The identification result of ∆pagP38 mutant, the result shows that pagP gene was deleted.
Mentions: Two suicide plasmids pQK001 and pQK002 were constructed to facilitate deletion of msbB and pagP gene in the BL21 (DE3), respectively, and the gene deletions were performed by homologous recombination via SacB-based counter-selection in allelic-exchange experiments [37]. Figure 1 illustrates the chromosomal structures of ∆msbB28 and ∆pagP38 in BL21 (DE3), and their PCR product size when amplified by flanking primers. The intermediate single ∆msbB28 or ∆pagP38 mutants were further confirmed by DNA sequencing. ∆pagP38 was introduced into intermediate mutant ∆msbB28 to yield the mutant S001 (∆msbB28 ∆pagP38), which was hypothesized to result in production of penta-acylated lipid A in this progeny mutant strain (Table 2).

Bottom Line: In this study, we constructed BL21 (DE3) ∆msbB28 ∆pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity.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 Article: PubMed Central - PubMed

Affiliation: Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China. p19890528@126.com.

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.

No MeSH data available.


Related in: MedlinePlus