Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

Abstract

When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in Escherichia coli, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g., trxB gor mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of trxB gor cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in E. coli required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.

Engineered Bacterial Outer Membrane Vesicles with Enhanced Functionality

“…Cytoplasmic and periplasmic fractions from cells expressing fusion proteins were generated by the cold osmotic shock procedure 53 and the pellet remaining after removal of the soluble fraction was collected as the insoluble fraction.…”

Production of Secretory and Extracellular N-Linked Glycoproteins inEscherichia coli

“…The supernatant fraction was subjected to 0.2-m filtration and then concentrated with an Amicon Ultra centrifugal filter from Millipore. The cell pellet was washed and then subjected to subcellular fractionation into periplasmic and cytoplasmic fractions using the ice-cold osmotic-shock procedure as described elsewhere (13,31). Outer membrane vesicles (OMVs) were isolated from cell-free supernatants as described previously (58).…”

Modified penicillin acylase signal peptide allows the periplasmic production of soluble human interferon-γ but not of soluble human interleukin-2 by the Tat pathway in Escherichia coli

“…It has been reported that the use of low post-induction temperatures (20-288C) increases solubility of the recombinant proteins transported to periplasm of E. coli (Robbens et al 2006). Kim et al (2005) reported the TorA signal peptide is capable of transporting complex eukaryotic proteins such as the human tissue plasminogen activator to the bacterial periplasm by the Tat pathway. In the case of hIL-2, the processed protein was detected in both, the CS and IM fractions at 9.8% and 7.3%, respectively, and as precursor in the CS and IM fractions at 3.6% and 79.3%, respectively (Fig.…”

“…First, the Tat pathway can transport complex proteins containing FeS, Ni-Fe, molybdopterin center (Berks 1996;Palmer et al 2005), heme group (Sturm et al 2006) and multidisulfide bonds (Kim et al 2005). Second, under aerobic conditions, few proteins use the Tat pathway, making this system readily available (Robinson and Bolhuis 2004).…”