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Co-solvents as stabilizing agents during heterologous overexpression in Escherichia coli - application to chlamydial penicillin-binding protein 6.

Otten C, De Benedetti S, Gaballah A, Bühl H, Klöckner A, Brauner J, Sahl HG, Henrichfreise B - PLoS ONE (2015)

Bottom Line: Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies.To solve this problem use of chaperones or in vitro refolding procedures are the means of choice.Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."

View Article: PubMed Central - PubMed

Affiliation: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.

ABSTRACT
Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."

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In vitro activity of PBP6Cp.The purified enzyme showed DD-carboxypeptidase activity on lipid II. (a, c) TLC and (b) MS analysis of reaction products. Cleaving of terminal D-Ala from the pentapeptide side chain of lipid II resulted in the formation of undecaprenyl-pyrophosphoryl-MurNAc-(GlcNAc)-tetrapeptide. (a,b) The exchange of S60 in the SxxK motif as well as (c) inhibition by penicillin G lead to a loss of function. CA: clavulanic acid; Pen: penicillin G.
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pone.0122110.g003: In vitro activity of PBP6Cp.The purified enzyme showed DD-carboxypeptidase activity on lipid II. (a, c) TLC and (b) MS analysis of reaction products. Cleaving of terminal D-Ala from the pentapeptide side chain of lipid II resulted in the formation of undecaprenyl-pyrophosphoryl-MurNAc-(GlcNAc)-tetrapeptide. (a,b) The exchange of S60 in the SxxK motif as well as (c) inhibition by penicillin G lead to a loss of function. CA: clavulanic acid; Pen: penicillin G.

Mentions: Chlamydiaceae genomes encode only three PBPs [8]. Being homologs of PBP2 and PBP3 from E. coli, the two high-molecular-weight PBPs are predicted to serve as monofunctional transpeptidases which cross-link adjacent peptidoglycan strands in E. coli [8]. The only low-molecular-weight PBP is classified as a class C type 5 PBP having 29% amino acid sequence identity with E. coli PBP6. In conventional bacteria that are surrounded by a classical cell wall envelope, these enzymes function as DD-carboxypeptidases and modulate the mature peptidoglycan meshwork by cleaving terminal D-Ala from pentapeptide side chains that are not cross-linked [18]. After successful betaine-assisted overexpression and purification of soluble PBP6Cp, the protein was tested for in vitro activity. In the absence of a cell wall envelope in Chlamydiaceae, we tested the ability to cleave terminal D-Ala from the completed cell wall building block lipid II. Prior to the experiment, PBP6Cp was rebuffered in co-solvent free buffer to avoid possible betaine-related effects on downstream reaction product detection. Thin layer chromatography (TLC) analysis as well as mass spectrometry revealed DD-carboxypeptidase activity on lipid II implicating that PBP6Cp was overexpressed and purified in its active, correctly-folded form (Fig 3a and 3b).


Co-solvents as stabilizing agents during heterologous overexpression in Escherichia coli - application to chlamydial penicillin-binding protein 6.

Otten C, De Benedetti S, Gaballah A, Bühl H, Klöckner A, Brauner J, Sahl HG, Henrichfreise B - PLoS ONE (2015)

In vitro activity of PBP6Cp.The purified enzyme showed DD-carboxypeptidase activity on lipid II. (a, c) TLC and (b) MS analysis of reaction products. Cleaving of terminal D-Ala from the pentapeptide side chain of lipid II resulted in the formation of undecaprenyl-pyrophosphoryl-MurNAc-(GlcNAc)-tetrapeptide. (a,b) The exchange of S60 in the SxxK motif as well as (c) inhibition by penicillin G lead to a loss of function. CA: clavulanic acid; Pen: penicillin G.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4388811&req=5

pone.0122110.g003: In vitro activity of PBP6Cp.The purified enzyme showed DD-carboxypeptidase activity on lipid II. (a, c) TLC and (b) MS analysis of reaction products. Cleaving of terminal D-Ala from the pentapeptide side chain of lipid II resulted in the formation of undecaprenyl-pyrophosphoryl-MurNAc-(GlcNAc)-tetrapeptide. (a,b) The exchange of S60 in the SxxK motif as well as (c) inhibition by penicillin G lead to a loss of function. CA: clavulanic acid; Pen: penicillin G.
Mentions: Chlamydiaceae genomes encode only three PBPs [8]. Being homologs of PBP2 and PBP3 from E. coli, the two high-molecular-weight PBPs are predicted to serve as monofunctional transpeptidases which cross-link adjacent peptidoglycan strands in E. coli [8]. The only low-molecular-weight PBP is classified as a class C type 5 PBP having 29% amino acid sequence identity with E. coli PBP6. In conventional bacteria that are surrounded by a classical cell wall envelope, these enzymes function as DD-carboxypeptidases and modulate the mature peptidoglycan meshwork by cleaving terminal D-Ala from pentapeptide side chains that are not cross-linked [18]. After successful betaine-assisted overexpression and purification of soluble PBP6Cp, the protein was tested for in vitro activity. In the absence of a cell wall envelope in Chlamydiaceae, we tested the ability to cleave terminal D-Ala from the completed cell wall building block lipid II. Prior to the experiment, PBP6Cp was rebuffered in co-solvent free buffer to avoid possible betaine-related effects on downstream reaction product detection. Thin layer chromatography (TLC) analysis as well as mass spectrometry revealed DD-carboxypeptidase activity on lipid II implicating that PBP6Cp was overexpressed and purified in its active, correctly-folded form (Fig 3a and 3b).

Bottom Line: Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies.To solve this problem use of chaperones or in vitro refolding procedures are the means of choice.Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."

View Article: PubMed Central - PubMed

Affiliation: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.

ABSTRACT
Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."

Show MeSH
Related in: MedlinePlus