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

<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

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

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

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

Catalytic Cycle of the <i>N</i>‑Acetylglucosaminidase NagZ from <i>Pseudomonas aeruginosa</i>

The <i>N</i>-acetylglucosaminidase NagZ of Pseudomonas aeruginosa catalyzes the first cytoplasmic step in recycling of muropeptides, cell-wall-derived natural products. This reaction regulates gene expression for the β-lactam resistance enzyme, β-lactamase. The enzyme catalyzes hydrolysis of <i>N</i>-acetyl-β-d-glucosamine-(1→4)-1,6-anhydro-<i>N</i>-acetyl-β-d-muramyl-peptide (<b>1</b>) to <i>N</i>-acetyl-β-d-glucosamine (<b>2</b>) and 1,6-anhydro-<i>N</i>-acetyl-β-d-muramyl-peptide (<b>3</b>). The structural and functional aspects of catalysis by NagZ were investigated by a total of seven X-ray structures, three computational models based on the X-ray structures, molecular-dynamics simulations and mutagenesis. The structural insights came from the unbound state and complexes of NagZ with the substrate, products and a mimetic of the transient oxocarbenium species, which were prepared by synthesis. The mechanism involves a histidine as acid/base catalyst, which is unique for glycosidases. The turnover process utilizes covalent modification of D244, requiring two transition-state species and is regulated by coordination with a zinc ion. The analysis provides a seamless continuum for the catalytic cycle, incorporating large motions by four loops that surround the active site