Limits...
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

The schematic structure of lipid A. The number of phosphate and fatty acyl chain of lipid A is indicated and the spectrum number m/z of lipid A structure is shown. The deletion of msbB and overexpression of pagL could change the number of fatty acyl chains of lipid A. Expression of lpxE in E. coli could remove the phosphate group of lipid A. Both structural modifications therefore change lipid A to tetra-acylated monophosphoryl lipid A.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4483635&req=5

marinedrugs-13-03388-f003: The schematic structure of lipid A. The number of phosphate and fatty acyl chain of lipid A is indicated and the spectrum number m/z of lipid A structure is shown. The deletion of msbB and overexpression of pagL could change the number of fatty acyl chains of lipid A. Expression of lpxE in E. coli could remove the phosphate group of lipid A. Both structural modifications therefore change lipid A to tetra-acylated monophosphoryl lipid A.

Mentions: Lipid A were extracted from BL21(DE3) and its derivatives by the Bligh-Dyer method [44], and analyzed by MALDI-TOF-MS (Figure 3 and Figure 4). The lipid A isolated from parent strain BL21(DE3) contains a predominant hexa-acylated lipid A (m/z 1796.2 in Figure 4) and a minor peak of hexa-acylated lipid A decorated with a 4-amino-4-deoxy-l-arabinose (l-Ara4N) residue (m/z 1950.1), which is consistent with other reports [45]. S001 (∆msbB28 ∆pagP38) yielded a major peak at m/z 1586.3 in the spectrum, resulting from failure of myristic acid chain addition to the 3′-position of lipid A due to the msbB deletion (Figure 3 and Figure 4B), and there was the second major peak at m/z 1739.5, which was a penta-acylated lipid A modified with l-Ara4N. As expected, PagL expression in the strain S002 lead to a major peak of 3-O-deacylated lipid A at m/z 1359.8 in the spectrum, which is tetra-acylated lipid A (Figure 3 and Figure 4C), and LpxE expression resulted in production of mono-phosphorylpenta-acylated lipid A (m/z 1506.1) and a minor peak at m/z 1629.1 which is a mono-phosphorylpenta-acylated lipid A derivative of a phosphoethanolamine (pEtN) addition to the 4′-phosphate position in the strain S003 (Figure 4D); in the spectrum of lipid A isolated from the strain S004 with both PagL and LpxE expression, there are two major peaks at m/z 1279.9 and m/z 1402.9 and a minor peak at m/z 1506.1; the peak at m/z 1279.9 is a dephosphorylated tetra-acylated lipid A at the 1-position, and a pEtN addition to dephosphorylated tetra-acylated lipid A at the 4′-position lead to the peak at m/z 1402.9, and the peak at m/z 1506.1 is a mono-phosphorylpenta-acylated lipid A, indicating that PagL is not fully functional in the strain S004.


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)

The schematic structure of lipid A. The number of phosphate and fatty acyl chain of lipid A is indicated and the spectrum number m/z of lipid A structure is shown. The deletion of msbB and overexpression of pagL could change the number of fatty acyl chains of lipid A. Expression of lpxE in E. coli could remove the phosphate group of lipid A. Both structural modifications therefore change lipid A to tetra-acylated monophosphoryl lipid A.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-03388-f003: The schematic structure of lipid A. The number of phosphate and fatty acyl chain of lipid A is indicated and the spectrum number m/z of lipid A structure is shown. The deletion of msbB and overexpression of pagL could change the number of fatty acyl chains of lipid A. Expression of lpxE in E. coli could remove the phosphate group of lipid A. Both structural modifications therefore change lipid A to tetra-acylated monophosphoryl lipid A.
Mentions: Lipid A were extracted from BL21(DE3) and its derivatives by the Bligh-Dyer method [44], and analyzed by MALDI-TOF-MS (Figure 3 and Figure 4). The lipid A isolated from parent strain BL21(DE3) contains a predominant hexa-acylated lipid A (m/z 1796.2 in Figure 4) and a minor peak of hexa-acylated lipid A decorated with a 4-amino-4-deoxy-l-arabinose (l-Ara4N) residue (m/z 1950.1), which is consistent with other reports [45]. S001 (∆msbB28 ∆pagP38) yielded a major peak at m/z 1586.3 in the spectrum, resulting from failure of myristic acid chain addition to the 3′-position of lipid A due to the msbB deletion (Figure 3 and Figure 4B), and there was the second major peak at m/z 1739.5, which was a penta-acylated lipid A modified with l-Ara4N. As expected, PagL expression in the strain S002 lead to a major peak of 3-O-deacylated lipid A at m/z 1359.8 in the spectrum, which is tetra-acylated lipid A (Figure 3 and Figure 4C), and LpxE expression resulted in production of mono-phosphorylpenta-acylated lipid A (m/z 1506.1) and a minor peak at m/z 1629.1 which is a mono-phosphorylpenta-acylated lipid A derivative of a phosphoethanolamine (pEtN) addition to the 4′-phosphate position in the strain S003 (Figure 4D); in the spectrum of lipid A isolated from the strain S004 with both PagL and LpxE expression, there are two major peaks at m/z 1279.9 and m/z 1402.9 and a minor peak at m/z 1506.1; the peak at m/z 1279.9 is a dephosphorylated tetra-acylated lipid A at the 1-position, and a pEtN addition to dephosphorylated tetra-acylated lipid A at the 4′-position lead to the peak at m/z 1402.9, and the peak at m/z 1506.1 is a mono-phosphorylpenta-acylated lipid A, indicating that PagL is not fully functional in the strain S004.

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