Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli

Abstract

Bacteria are simple and cost effective hosts for producing recombinant proteins. However, their physiological features may limit their use for obtaining in native form proteins of some specific structural classes, such as for instance polypeptides that undergo extensive post-translational modifications. To some extent, also the production of proteins that depending on disulfide bridges for their stability has been considered difficult in E. coli.Both eukaryotic and prokaryotic organisms keep their cytoplasm reduced and, consequently, disulfide bond formation is impaired in this subcellular compartment. Disulfide bridges can stabilize protein structure and are often present in high abundance in secreted proteins. In eukaryotic cells such bonds are formed in the oxidizing environment of endoplasmic reticulum during the export process. Bacteria do not possess a similar specialized subcellular compartment, but they have both export systems and enzymatic activities aimed at the formation and at the quality control of disulfide bonds in the oxidizing periplasm.This article reviews the available strategies for exploiting the physiological mechanisms of bactera to produce properly folded disulfide-bonded proteins.

Figure 1

Schematic representation of folding pathways and cellular localization of proteins that depend on oxidative environment to reach their native structure. Unfolded proteins can be translocated into the periplasm post-translationally (Sec mechanism) or co-translationally (SPR mechanism), according to their specific leader sequences. SecB is the physiological chaperone involved in stabilizing unfolded proteins bound to Sec translocation machinery, but the activity of other cytoplasmic chaperones like DnaK can be beneficial. Once in the periplasm proteins folding is mediated by the Dsb oxidases/isomerases, by chaperones such as Skp, DegP and FkpA, and by peptidyl-prolyl isomerases such as SurA, PpiA, and PpiB. Double mutant strains, in which the thioredoxin/glutaredoxin reductase (trxB^-, gor^-) pathway is silenced, are characterized by the presence of an oxidizing cytoplasm, compatible with the folding of proteins that need disulfide bridges for stabilizing their structure. Cytoplasmic accumulation of correctly folded disulfide-dependent proteins is improved by disulfide isomerase DsbC co-expression and cytoplasmic chaperone (DnaKJE, GroELS, ClpB). Finally, proteins provided with a Tat export leader sequence first fold in the cytoplasm and then are tanslocated into the periplasm. Chaperones like DnaKJE, can stabilize the precursor in the cytoplasm and chaperones such as SlyD and TorD support the pathway efficiency probably binding to the export leader peptide.

Figure 2

Flow-chart summarizing the different possibilities for producing disulfide-dependent proteins in bacteria. Expression is optimized and protein folding directed either in the cytoplasm or in the periplasm. Once folded in the cytoplasm, proteins can accumulate in the same cell compartment, or be exported to the periplasm by the Tat-pathway, or be secreted to the medium by the type I secretion system. Unfolded proteins can be translocated post-translationaly (Sec) or co-translationaly (SPR) into the periplasm and accumulate there, leak to the medium, or be exposed on the outer membrane. Precipitated proteins can be recovered into native structure by means of oxidative refolding.

Figure 3

Dsb-dependent protein oxidation and isomerization in the bacterial periplasm. DsbA couples consecutive cysteines providing the disulfide bond and is re-charged by the inner membrane DsbB. Incorrect disulfides of oxidized proteins are scrambled by DsbC and DsbG. These isomerases are kept reduced by the inner membrane DsbD that, in return, is reduced by cytoplasmic thioredoxin.