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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 plasmids harboring pagL and/or lpxE were then introduced into this mutant to further modify the LPS.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

Plasmid maps used in this study. Maps of three expression plasmids constructed in this study. Plasmid pQK003 was constructed for expressing pagL to decrease the fatty acyl chain number. Plasmid pQK004 was constructed for expressing lpxE to remove 1-phosphate group in lipid A. Plasmid pQK005 was constructed for expressing pagL and lpxE to confer both of these functions.
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marinedrugs-13-03388-f002: Plasmid maps used in this study. Maps of three expression plasmids constructed in this study. Plasmid pQK003 was constructed for expressing pagL to decrease the fatty acyl chain number. Plasmid pQK004 was constructed for expressing lpxE to remove 1-phosphate group in lipid A. Plasmid pQK005 was constructed for expressing pagL and lpxE to confer both of these functions.

Mentions: The other three plasmids were constructed for overexpressing pagL (pQK003), lpxE (pQK004), and both pagI and IpxE (pQK005) under constitutive transcriptional control of the strong E. coli promoter Plpp (Figure 2). These plasmids replicate with low copy number in E. coli (origin of replication p15a) and encode a Cm resistance marker to facilitate use in other bacterial strains. Plasmids pQK003, pQK004 and pQK005 were transformed separately to S001 to yield the strains S002, S003 and S004, respectively (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)

Plasmid maps used in this study. Maps of three expression plasmids constructed in this study. Plasmid pQK003 was constructed for expressing pagL to decrease the fatty acyl chain number. Plasmid pQK004 was constructed for expressing lpxE to remove 1-phosphate group in lipid A. Plasmid pQK005 was constructed for expressing pagL and lpxE to confer both of these functions.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-03388-f002: Plasmid maps used in this study. Maps of three expression plasmids constructed in this study. Plasmid pQK003 was constructed for expressing pagL to decrease the fatty acyl chain number. Plasmid pQK004 was constructed for expressing lpxE to remove 1-phosphate group in lipid A. Plasmid pQK005 was constructed for expressing pagL and lpxE to confer both of these functions.
Mentions: The other three plasmids were constructed for overexpressing pagL (pQK003), lpxE (pQK004), and both pagI and IpxE (pQK005) under constitutive transcriptional control of the strong E. coli promoter Plpp (Figure 2). These plasmids replicate with low copy number in E. coli (origin of replication p15a) and encode a Cm resistance marker to facilitate use in other bacterial strains. Plasmids pQK003, pQK004 and pQK005 were transformed separately to S001 to yield the strains S002, S003 and S004, respectively (Table 2).

Bottom Line: 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.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