Efficient export of prefolded, disulfide‐bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains

Abstract

Numerous high-value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide-containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild-type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide-bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm.

Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation

“…However, some proteins were exported in an E. coli mutant strain, Δgor/ΔtrxB, that allows formation of the disulfide bonds in the cytoplasm [5]. Similar results were obtained by our group when we expressed disulfide-requiring proteins in 'CyDisCo' (Cytoplasmic Disulfide formation in E. coli) strains that promote efficient formation of disulfide bonds in the cytoplasm [16]. The Tat pathway thus clearly identified these constructs as misfolded in wild type strains and rejected them.…”

“…Previous studies [5,16] showed that several disulfide-bonded proteins, including PhoA, a phytase AppA, an scFv construct and a Fab fragment, were exported in E. coli when a Tat signal peptide was present at the N-terminus, provided that disulfide formation could occur in the cytoplasm. In one case this was achieved by expression in Δgor/ΔtrxB cells; this strain passively enables the formation of disulfide bonds in proteins in the cytoplasm by the removal of the two naturally occurring reducing pathways [23].…”

“…The product was then digested with NdeI and BamHI (New England Biolabs) and inserted into pYU49 which had been cut similarly [16].…”

“…Several proteins which contain disulfide bonds in the native state could not be exported by Tat, because they were found to be misfolded, and hence completely Tat-incompatible, in the absence of cytoplasmic disulfide formation [5,16]. However, some proteins were exported in an E. coli mutant strain, Δgor/ΔtrxB, that allows formation of the disulfide bonds in the cytoplasm [5].…”

“…An example is green fluorescent protein (GFP), a tightly-folding protein that cannot normally be exported by Sec, but which is very efficiently exported by Tat [27]. Moreover, the Tat pathway can support levels of protein export that are comparable to those obtained in Sec-dependent industrial production systems; fed-batch fermentation studies showed that periplasmic GFP levels exceeded 1 g protein per litre culture [16].…”