Exploration of twin‐arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

Abstract

SummaryHistorically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step.

Production of Secretory and Extracellular N-Linked Glycoproteins inEscherichia coli

“…The molecular mass of this band was just under 50 kDa, which likely corresponds to tetraglycosylated AcrA-4ϫ (44.2 kDa) that has retained its Nterminal spTorA signal peptide (4.2 kDa). Indeed, it has been observed that overexpression of Tat substrates and cell stress negatively affect the efficiency of signal peptide removal in E. coli (19,54). Based on the slow electrophoretic mobility of this protein and its analysis by nanoLC-MS/MS, which revealed N-linked glycosylation at all four sites (data not shown), we conclude that tetraglycosylated AcrA-4ϫ is the predominant glycosylated species produced by the Tat pathway.…”

High‐level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin‐arginine translocation system in Escherichia coli

“…a large scale. It transports folded proteins across the plasma membrane, and even appears to preferentially transport correctly folded proteins [5,6]. It may therefore be particularly advantageous for the export of Secincompatible proteins.…”

“…Importantly, the Tat system translocates fully folded proteins, and it thus differs in fundamental respects from the Sec pathway. The system has been shown to be able to transport a variety of heterologous proteins [4][5][6].…”

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

“…For example, in the case of MBP, after formation of an ssTorA-X::Y-MBP complex, MBP must bind maltose and then deliver it to the MalFGK(2) inner membrane complex. The intricacy of this reporter activity may prove problematic, especially considering that, in certain instances, direct fusions to MBP (e.g., ssTorA-GFP-MBP) are incapable of performing this function even when the fusion protein is correctly localized to the periplasm and the fusion partner (e.g., GFP) is active (52). It should also be pointed out that even though previous reports suggest Bla is undesirable as a reporter because low-level resistance to Amp has been observed with Bla lacking a signal peptide (33,51), in our hands, we never observed nonspecific leakage of Bla to the periplasm (Fig.…”