Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm

Abstract

Under physiological conditions, the Escherichia coli cytoplasm is maintained in a reduced state that strongly disfavors the formation of stable disulfide bonds in proteins. However, mutants in which the reduction of both thioredoxins and glutathione is impaired (trxB gor mutants) accumulate oxidized, enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate extragenic suppressors at a high frequency. One such suppressor strain, FA113, grows almost as rapidly as the wild type in the absence of reductant, exhibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substantially higher yields of properly oxidized proteins compared with wild-type or trxB mutant strains. For polypeptides with very complex patterns of disulfide bonds, such as vtPA and the full-length tPA, the amount of active protein was further enhanced up to 15-fold by co-expression of TrxA (thioredoxin 1) mutants with different redox potentials, or 20-fold by the protein disulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of E. coli FA113 compared with what could be achieved via secretion into the periplasm of a wild-type strain, even under optimized conditions. These results demonstrate that the cytoplasm can be rendered sufficiently oxidizing to allow efficient formation of native disulfide bonds without compromising cell viability.

Figure 1

tPA activity in DHB4 (wild type), WP597 (trxB), FA112 (trxB gshA supp), and FA113 (trxB gor supp) monitored by the fibrinolysis assay. Cells were transformed with plasmids pTrcvtPA, and pFA5 (bottom row only). Soluble protein (10 μg) from induced cultures was spotted onto fibrin/agarose plates and was incubated for 24 hr at 37°C. Clearance zones qualitatively measure biological activity of bacterially produced vtPA.

Figure 2

Growth curves of DHB4 (●); FA113 (■); FA113/pTrcvtPA/pFA5 (♦); and DHB4/pTrcStIIvtPA/pBADdsbC (▴) grown aerobically at 37°C in LB medium in test tubes. Expression of vtPA was induced at late log phase as described in the text. Optical density was measured in a microtiter plate reader.

Figure 3

Pulse–chase of alkaline phosphatase. Signal sequenceless alkaline phosphatase (AP) was immunoprecipitated and separated by native PAGE, such that the oxidized form (ox) was distinguished from the reduced form (red). Time is indicated in minutes postchase. Strains used were DHB4 (wild type), WP597 (trxB), and FA113 (trxB gor supp) transformed with plasmid pAID135. OmpA was used as an internal standard.

Figure 4

The effect of coexpression of vtPA and oxidoreductases in the cytoplasm of strain FA113 (trxB gor supp) quantified by an indirect assay for plasminogen activation using a chromogenic plasmin substrate (see text). Activity has been normalized to the value obtained from vtPA expressed alone in FA113 (column 1). Cell lysates used for columns 2–8 were from strains cotransformed with plasmids pFA2-pFA8, respectively. Plasmids pFA3–8 overexpress active site mutants of TrxA. The amino acid sequence of the active site is listed, along with the known oxidoreductase that contains that native active site.

Figure 5

Plasminogen activation in lysates of cells expressing vtPA secreted to the periplasm of DHB4 (wild type) or in the cytoplasm of FA113 (trxB gor supp). Foldases were coexpressed from plasmids pBADdsbA, pBADdsbC, pBADΔSSdsbA, pBADΔSSdsbC, pFA3, and pFA5.