Co-Solvents as Stabilizing Agents during Heterologous Overexpression in <i>Escherichia coli</i> – Application to Chlamydial Penicillin-Binding Protein 6

<div><p>Heterologous overexpression of foreign proteins in <i>Escherichia coli</i> often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or <i>in vitro</i> 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 <i>in vitro</i> 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.”</p></div

Systematic use of co-solvents in heterologous overexpression and purification of proteins.

<p>(a) Workflow for the co-solvent assisted overproduction and purification method and (b) cartoon on the step of co-solvent assisted overexpression in the <i>E</i>. <i>coli</i> periplasm illustrating the mode of action of co-solvents.</p

<i>In vitro</i> activity of PBP6_Cp.

<p>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.</p

Most common co-solvents in biopharmaceutical formulation included in the co-solvent screen described in this study.

<p>*not used in this study</p><p>Most common co-solvents in biopharmaceutical formulation included in the co-solvent screen described in this study.</p

Energy levels of the denatured and native state of a protein.

<p>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.</p

Application of the co-solvent assisted method to PBP6_Cp.

<p>(a) Structure of PBP6_Cp. Domains, motifs, signal peptides (SP), transmembrane domains (TM), leader peptide sequence for transportation into the periplasm (OmpA) and chromatography affinity tag (Strep) are depicted (SP and TM were predicted by Signal P and TMHMM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122110#pone.0122110.ref030" target="_blank">30</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122110#pone.0122110.ref031" target="_blank">31</a>]. The modified PBP6_Cp (42.95 kDa) which lacks the native SP and TM domain was overproduced, purified and tested for DD-carboxypeptidase activity. (b) Results from overexpression pretest (western blot), (c) co-solvent screen western blot), and (d) betaine-assisted purification (Coomassie stain) of PBP6_Cp. The optimal expression conditions (<i>E</i>. <i>coli</i> C43(DE3), 4h of induction at 25°C) and co-solvent (betaine) determined in the overexpression pretest and co-solvent screen, respectively, were used in an up-scaled culture to overproduce soluble PBP6_Cp for the first time. Pre: pre induction, S: soluble fraction, P: pellet fraction, w/o: without the addition of co-solvent, Glc: glucose, Suc: sucrose, Tre: trehalose, Lac: lactose, Gly: glycerol, Man: mannitol, Sor: sorbitol, Bet: betaine, His: histidine. BCCP (21.5kDa): biotin carboxyl carrier protein from <i>E</i>. <i>coli</i>, a common contamination of strep-tagged proteins (biotinylated protein binding to strep-tactin which can be removed by complexation with avidin from hen egg white [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122110#pone.0122110.ref012" target="_blank">12</a>]).</p