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

Abstract

Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.

Proofreading of substrate structure by the Twin-Arginine Translocase is highly dependent on substrate conformational flexibility but surprisingly tolerant of surface charge and hydrophobicity changes

“…We further confirmed that the disulphide bonds remain reduced during the purification process; Supplementary data A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 12 We have previously shown that a different scFv could be exported in wild type or CyDisCo cells (i.e. in either the reduced or oxidised form; [23]); we therefore first tested whether the disulphide bonding status affects export of scFvM by Tat in these strains. The protein was expressed with a TorA signal peptide, and reveals that export is ca.…”

“…To this end we chose an scFv (termed scFvM, to distinguish it from the different scFv used by Alanen et al [23]) and we then generated and tested a range of mutants, including some sourced from a biophysical study of the scFv and mutants in solution (manuscript in preparation). The predicted structure scFvM, based on homology modelling with PDB file 2GHW, is shown in Fig.…”

“…The remaining images show the charge and hydrophobicity characterostics from two different projections. For tests on the Tat proofreading system, scFvM was expressed with an N-terminal TorA signal peptide and a Cterminal 6x His-tag, as used in a previous study on a different scFv [23].…”

“…For example, it was shown that several disulphide-bonded proteins (including human growth hormone, a single-chain variable fragment (scFv) and interferon α2β) can be exported in the reduced state in wild type cells [23] and the native Tat substrate CueO is exported in its apo form [24]. However, these proteins are believed to adopt near-native structures even in the absence of disulphide bond formation or Cu ligation, and so the extent of misfolding is likely to be minimal.…”

Generation of Recombinant N-Linked Glycoproteins in E. coli

“…PglB, as highlighted previously, is a membrane bound protein located in the periplasm, so is already present. As for the target protein, there are multiple pathways available that will direct the target protein to this part of the cell, including the TAT export system [40], SRP pathway [41], and sec transport system [42]. The most utilized methodology is the sec transport system, which requires the addition of a 22 amino acid leader sequence at the N-terminus of the polypeptide chain.…”