title: High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli.
abstract: Numerous high-value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin-arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed-batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial-type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA-GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co-expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over-expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed-batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h(-1), the cultures reached OD(600) values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L(-1) culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec-dependent export approaches.
authors.affiliation: School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Cristina F R O
authors.initials: CF
authors.lastname: Matos
authors.forename: Steven D
authors.initials: SD
authors.lastname: Branston
authors.forename: Anna
authors.initials: A
authors.lastname: Albiniak
authors.forename: Arjun
authors.initials: A
authors.lastname: Dhanoya
authors.forename: Robert B
authors.initials: RB
authors.lastname: Freedman
authors.forename: Eli
authors.initials: E
authors.lastname: Keshavarz-Moore
authors.forename: Colin
authors.initials: C
authors.lastname: Robinson
mesh_terms: D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D019897:Periplasm / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C408585:twin-arginine translocase complex, E coli; D049452:Green Fluorescent Proteins