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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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Related in: MedlinePlus

Energy levels of the denatured and native state of a protein.In the diagram ΔG is representing the free energy necessary to unfold the protein. In case 1, upon addition the co-solvent is excluded from the surface of the denatured state of the protein and by that increasing the energy level of the denatured state. In case 2, the co-solvent only binds to the native state of the protein and lowers the energy level. Case 3 illustrates the mode of action of most co-solvents to stabilize proteins. Exclusion of the co-solvent from both, the native and the denatured state, leads to an overall increased level of free energy. The green and the orange shape represent the protein in its native and denatured state, respectively.
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pone.0122110.g004: Energy levels of the denatured and native state of a protein.In the diagram ΔG is representing the free energy necessary to unfold the protein. In case 1, upon addition the co-solvent is excluded from the surface of the denatured state of the protein and by that increasing the energy level of the denatured state. In case 2, the co-solvent only binds to the native state of the protein and lowers the energy level. Case 3 illustrates the mode of action of most co-solvents to stabilize proteins. Exclusion of the co-solvent from both, the native and the denatured state, leads to an overall increased level of free energy. The green and the orange shape represent the protein in its native and denatured state, respectively.

Mentions: To characterize the stabilizing effects of co-solvents on a thermodynamic level Wyman stated the following linked function: (δ ln K / δ ln ax)T, P, a = νx Prod—νx React = Δνx (K = equilibrium constant, ν = binding of the ligand) [23]. It describes the influence of a substance (x) on either the native or the denatured state of a protein in dependency of the concentration (activity, a), temperature (T) and pressure (P). Based on the work of Wyman, Timasheff developed a model that describes the influence of co-solvents by showing the interactions between the co-solvent and the protein [5]. In general, denaturation of a protein requires a certain change in free energy (ΔG), whereby the cold denaturation represents a special case with a change in heat capacity [24]. To stabilize a protein you can lower the level of free energy of the native state (case 2), increase the level for the denatured state (case 1) or increase the overall energy level (case 3) (Fig 4). The latter case represents the underlying mechanism of “preferential exclusion” and the mode of action of most co-solvents including all substances used in our screen (Table 1).


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)

Energy levels of the denatured and native state of a protein.In the diagram ΔG is representing the free energy necessary to unfold the protein. In case 1, upon addition the co-solvent is excluded from the surface of the denatured state of the protein and by that increasing the energy level of the denatured state. In case 2, the co-solvent only binds to the native state of the protein and lowers the energy level. Case 3 illustrates the mode of action of most co-solvents to stabilize proteins. Exclusion of the co-solvent from both, the native and the denatured state, leads to an overall increased level of free energy. The green and the orange shape represent the protein in its native and denatured state, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122110.g004: Energy levels of the denatured and native state of a protein.In the diagram ΔG is representing the free energy necessary to unfold the protein. In case 1, upon addition the co-solvent is excluded from the surface of the denatured state of the protein and by that increasing the energy level of the denatured state. In case 2, the co-solvent only binds to the native state of the protein and lowers the energy level. Case 3 illustrates the mode of action of most co-solvents to stabilize proteins. Exclusion of the co-solvent from both, the native and the denatured state, leads to an overall increased level of free energy. The green and the orange shape represent the protein in its native and denatured state, respectively.
Mentions: To characterize the stabilizing effects of co-solvents on a thermodynamic level Wyman stated the following linked function: (δ ln K / δ ln ax)T, P, a = νx Prod—νx React = Δνx (K = equilibrium constant, ν = binding of the ligand) [23]. It describes the influence of a substance (x) on either the native or the denatured state of a protein in dependency of the concentration (activity, a), temperature (T) and pressure (P). Based on the work of Wyman, Timasheff developed a model that describes the influence of co-solvents by showing the interactions between the co-solvent and the protein [5]. In general, denaturation of a protein requires a certain change in free energy (ΔG), whereby the cold denaturation represents a special case with a change in heat capacity [24]. To stabilize a protein you can lower the level of free energy of the native state (case 2), increase the level for the denatured state (case 1) or increase the overall energy level (case 3) (Fig 4). The latter case represents the underlying mechanism of “preferential exclusion” and the mode of action of most co-solvents including all substances used in our screen (Table 1).

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