Fig 1.
Systematic use of co-solvents in heterologous overexpression and purification of proteins.
(a) Workflow for the co-solvent assisted overproduction and purification method and (b) cartoon on the step of co-solvent assisted overexpression in the E. coli periplasm illustrating the mode of action of co-solvents.
Fig 2.
Application of the co-solvent assisted method to PBP6Cp.
(a) Structure of PBP6Cp. 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 [30,31]. The modified PBP6Cp (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 PBP6Cp. The optimal expression conditions (E. coli 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 PBP6Cp 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 E. coli, 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 [12]).
Table 1.
Most common co-solvents in biopharmaceutical formulation included in the co-solvent screen described in this study.
Fig 3.
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.
Fig 4.
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.