Ligand supplementation as a method to increase soluble heterologous protein production

Figures & data

Figure 1. Ligand supplementation (100 µM CBX during induction phase) leads to increased soluble expression of distinct human short-chain dehydrogenases/reductases, shown here by expression of type 11β-hydroxysteroid dehydrogenase (HSD).

(A) Different Escherichia coli strains (BL21 with and without a co-expression plasmid harboring the chaperone GroEL/ES [GroE7]) were analyzed for soluble expression by SDS-PAGE (left lane for each construct is soluble homogenate and the right is the eluate from immobilized metal-affinity chromatography purification). Two different constructs (denoted G25 and G26, with different domain boundaries of human 11β-HSD1) were used to inoculate the cultures. After initial incubation, cultures were split and treated with CBX or solvent (DMSO). Arrow indicates the expected mass of 11β-HSD1. (B) Ligand supplementation of human 11β-HSD1 with distinct chemical scaffolds (cpd 1, cpd 2, 18α-GA and 18β-GA). Different 11β-HSD1 inhibitors were used in analogous expression experiments, as presented for CBX (A). Note the specificity for the GA isomers 18α-GA and 18β-GA.

CBX: Carbenoxolone; ctrl: Control; GA: Glycerrhitinic acid.

Figure 2. Schematic representation of the ligand supplementation concept to enhance soluble protein production in Escherichia coli.

Nascent polypeptides can fold into apoproteins or aggregate and are then deposited in inclusion bodies. Inclusion of a ligand can promote and stabilize the folded protein, leading to a stable and soluble protein–ligand complex.