title: High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli.
abstract: The twin-arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat-type signal peptide is present at the N-terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA-GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16-h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large-scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L⁻¹ culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing.
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, UK.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Albiniak
authors.forename: Cristina F R O
authors.initials: CF
authors.lastname: Matos
authors.forename: Steven D
authors.initials: SD
authors.lastname: Branston
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: D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D061249:Batch Cell Culture Techniques; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D005285:Fermentation; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase
keywords: Bacillus subtilis TatAdCd; Escherichia coli; green fluorescent protein (GFP); twin arginine translocation (Tat) signal peptide