Coexpression of TorD enhances the transport of GFP via the TAT pathway

Abstract

Twin-arginine translocation (Tat) pathway is capable of secreting fully folded proteins into the periplasm of Gram-negative bacteria and may thus be an ideal system for the expression of active cofactor-containing proteins. However, the applications of Tat system for such purpose have been plagued by low translocation efficiencies. In this study, we demonstrate that the coexpression of a soluble chaperone, TorD, in conjunction with the TorA signal peptide, the translocation efficiency of GFP can be enhanced by more than three-fold. The enhancement in translocation efficiency is believed to be a result of reduced proteolysis mediated by the binding of TorD toward the TorA signal peptide. We believe this approach can be further exploited for the expression and secretion of other heterologous proteins as well as traditional Tat substrate proteins.

Introduction

It is often desirable to secrete recombinant proteins into the periplasm of Gram-negative bacteria such as E. coli for the many intrinsic advantages inherited with the physiological environments of the periplasm, which may increase the yields of soluble, active proteins (Makrides, 1996). For examples, the formation of the necessary disulfide bond pairs can be promoted in the periplasm. The stability of secreted proteins can also be greatly enhanced due to the reduced proteolysis in the periplasm (Talmadge and Gilbert, 1982). Furthermore, the N-terminal authenticity of the secreted proteins can be better ensured as the signal peptides are cleaved in vivo during the translocation processes (Paetzel et al., 1998). The concomitant secretion of recombinant proteins during protein expression may alleviate the formation of protein inclusion bodies (Loo et al., 2002). The recovery and purification of secreted, periplasmic proteins can also be greatly simplified by selectively breaking the outer membrane without rupturing the cytoplasmic membrane. Furthermore, the secretion of enzymes into the periplasm can also be exploited for the development of whole-cell biocatalysts. Significant reduction in mass transfer resistance, and thus enhancement in the rates of biotransformation can be achieved by translocating the enzymes into the periplasm or onto the surface of outer membrane of E. coli (Wan et al., 2002).

The secretion of proteins in E. coli is generally carried out via the well-characterized Sec pathway (Pugsley, 1990). Recently, the twin-arginine translocation (Tat) pathway, an ATP-independent secretion system initially found in chloroplast thylakoid (Cline et al., 1992), has been identified in bacteria for the secretion of the periplasmic cofactor-containing proteins (Berks, 1996, Berks et al., 2000, Robinson and Bolhuis, 2004). Proteins secreted via the Tat pathway are guided by signal peptides exhibiting characteristic SRR×FLK motifs located at the boundary between the polar n-regions and the hydrophobic h-regions (Berks, 1996). The employment of the Tat pathway for protein secretion has enticed great research interests because of its capability in translocating multisubunit, cofactor-containing proteins in folded states. It has been shown that the translocation of disulfided proteins could also be achieved when a trx mutant E. coli with was employed (Bessette et al., 1999, DeLisa et al., 2003). These remarkable characteristics of the Tat pathway make it a promising alternative for protein secretion, when the Sec pathway is deemed unsuitable due to its deficiency in secreting multimeric, disulfided, or cofactor-containing proteins.

Nevertheless, the applicability of Tat system for protein secretion has been limited by its low translocation efficiency compared to that of the Sec system. Even at low expression levels the efficiencies of protein translocation via the Tat pathway rarely reaches above 50% (Mikhaleva et al., 1999, Thomas et al., 2001, DeLisa et al., 2002, DeLisa et al., 2003, Stanley et al., 2002, Chanal et al., 2003). This phenomenon warrants further studies aiming for the enhancement of translocation efficiency via the Tat pathway. To this end, enhanced protein translocation by overexpressing Tat machinery or coexpressing phage shock protein A (PspA) has been reported (Barrett et al., 2003, DeLisa et al., 2004).

The Tat system's remarkable capability in selectively exporting folded, multimeric, cofactor-inserted proteins has brought up the question of how the proofreading process is carried out to prevent the premature translocation of unfolded or pre-assembled apoproteins. Among the several models proposed for such proofreading mechanism (Santini et al., 1998, Sargent et al., 2002, Bruser and Sanders, 2003, DeLisa et al., 2003, Dubini and Sargent, 2003, Jack et al., 2004, Palmer et al., 2005), the chaperone-assisted quality control model proposed by Sargent and co-workers is the most noteworthy (Dubini and Sargent, 2003, Jack et al., 2004, Palmer et al., 2005). Based on the model proposed, the Tat signal peptides operate in tandem with specific cellular chaperones in the insertion of cofactors, the oligomerization, and the translocation of complex proteins, providing a pre-export proofreading mechanism. By overexpressing TorD, enhanced Tat-mediated translocation of trimethylamino N-oxide (TAMO) reductase (TorA) and (NiFe) hydrogenase-2 with the TorA signal peptide was observed (Jack et al., 2004). It is believed that by its specific recognition of the signal peptide of TorA, TorD facilitates the loading of molybdenum cofactor and the oligomerization of protein subunits and prevents the premature translocation of the periplasmic redox enzymes.

The capability of TorD, in conjunction with TorA signal peptide, in facilitating and proofreading the translocation of seemingly unrelated hydrogenase-2 (Jack et al., 2004) has motivated us to investigate its role on the translocation of monomeric, cofactorless proteins. In this study we demonstrate that TorD enhances the translocation of GFP under the guidance of the TorA signal peptide, possibly by reducing the proteolysis of pre-GFP resulting from its specific binding to the TorA signal peptide.