Exploring the Tat pathway for the production of disulphide-bonded recombinant proteins using CyDisCo in Escherichia coli

The production of heterologous proteins of interest in microbial hosts such as Escherichia coli is a cost-effective and vital tool for the biopharmaceutical and enzyme industries. Research and development efforts over the past few decades have majorly focused on the Sec pathway for recombinant protein secretion in E. coli. However, the Twin-arginine translocation (Tat) pathway of E. coli has recently garnered interest for the periplasmic export of folded biopharmaceuticals as it possesses a unique proofreading ability to export correctly folded proteins. Tat-based translocation of biopharmaceuticals to the periplasm has the potential to greatly ease the downstream processing by reducing the amount of contaminating proteins and nucleic acids, thereby lowering the costs and time needed for their production. Furthermore, coupling Tatdependent protein secretion with the CyDisCo technology (cytoplasmic disulphide bond formation in E. coli), which enables disulphide bond formation in the cytoplasm of E. coli, provides a powerful platform for the production of industrially relevant and difficult-to-express proteins.

Initially, a new experimental design for the export of multiple disulphide bond containing proteins to the periplasm by Tat in the presence of CyDisCo was empirically tested and developed as a starting point for future experiments. Later, a comparative study between Sec and Tat pathways for periplasmic export of a single chain variable fragment (scFv) in E. coli BL21 and W3110 was carried out. This led to an interesting comparison between the two strains based on soluble protein yields and quality when exported by the Sec pathway in fermenters. We observed a stark difference in the productivity profile of these two strains and a similar heterogeneity profile in the final product in both strains that could not detected by mass spectrometry-based analysis but confirmed by Differential Scanning Calorimetry (DSC) and 1D-1 H -NMR spectroscopy. Finally, in order to understand the limitations and maximize the applicability of the Tat pathway for proteins that fail to get exported efficiently with the TorA signal peptide, we focused on investigating alternative signal peptides. Here, we found that alternative signal peptides namely AmiC and MdoD allow highly efficient secretion of a disulphide bond-containing protein (YebF) to the periplasm of E. coli via Tat with CyDisCo. We report that these signal peptides are far more efficient than the well-known Tat specific TorA signal peptide, and hence they are potentially more suitable for large scale protein production than this more rigorous Tat-specific signal peptide. Overall, the research work presented in this thesis suggests that the Tat pathway and the CyDisCo system are attractive platforms for biotechnology and establishes highly efficient Tatdependent secretion of disulphide-bonded protein YebF to the E. coli periplasm.