Versatile signal peptide of Flavobacterium‐originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli

Abstract

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016.

Secretion of recombinant proteins from E. coli

“…The addition of multiple TorA signal sequences can enhance the effect [145]. Examples for heterologous Tat-dependent signal sequences that can be used for recombinant protein expression are those of organophosphorus hydrolase (OPH) from Flavobacterium [150], Cel9B signal sequence from Ruminococcus albus major glycoside hydrolase [151], as well as DagA from Streptomyces coelicolor agarase [140]. Examples for heterologous Tat-dependent signal sequences that can be used for recombinant protein expression are those of organophosphorus hydrolase (OPH) from Flavobacterium [150], Cel9B signal sequence from Ruminococcus albus major glycoside hydrolase [151], as well as DagA from Streptomyces coelicolor agarase [140].…”

A novel variable antibody fragment dimerized by the dHLX peptide with enhanced affinity against amantadine compared to its corresponding scFv antibody

“…Producing scFvs at high levels in E. coli often results in the formation of inclusion bodies, it is common to encounter problems with solubility and proper folding (Worn & Plückthun, 2001), however in this study, the T7 promoter expression system is so efficient that mRNA synthesis is fast and directly coupled to translation, and the pelB signal peptide directs scFv secretion into the periplasmic space. which provide an oxidative environment suitable for functional proteins production and disulfide bond formation (Dreier & Plückthun, 2011;Jost & Plückthun, 2014;Kang et al, 2016;Plückthun 2015;Tamaskovic, Simon, Stefan, Schwill, & Plückthun, 2012). The oxidized environment of the periplasmic space, rich in disulfide bond formation proteins (PDI, DsbA and DsbC) and chaperones SKp (Lindner et al, 2014;Liu et al, 2008), is most suitable for natural folding of the scFv fragment with high yield.…”