Escherichia coli "TatExpress" strains export several g/L human growth hormone to the periplasm by the Tat pathway.

Isabel Guerrero Montero,Stephen J W Busby,Amber R Peswani,David P Humphreys,Cedric Aubry,Chillel Jawara,Matthew Allen,Colin Robinson,Mickael Labrit,Kirsty L Richards,Emma Davé,Douglas F Browning

doi:10.1002/bit.27147

Isabel Guerrero Montero, Stephen J W Busby + Show 10 more

Open Access

https://doi.org/10.1002/bit.27147

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Abstract

Escherichia coli is a heavily used platform for the production of biotherapeutic and other high‐value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed‐batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 “TatExpress” strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N‐terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed‐batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme‐linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two‐fold lower.

Highlights

Many high‐value proteins are produced in Escherichia coli, and a favored strategy is to export the protein to the periplasm, usually by the well‐characterized “Sec” pathway (Walsh, 2014)
We first expressed a construct comprising the TMAO reductase (TorA) signal peptide linked to human growth hormone (hGH) (TorA–hGH) in W3110 E. coli TatExpress cells under fed‐batch fermentation conditions as detailed in Materials and Methods
The Tat system has been proposed to offer a viable alternative to the Sec pathway for the export of high‐value proteins to the bacterial periplasm, but its potential for the production of biotherapeutic proteins has not been fully explored in previous studies

Summary

| INTRODUCTION

Many high‐value proteins are produced in Escherichia coli, and a favored strategy is to export the protein to the periplasm, usually by the well‐characterized “Sec” pathway (Walsh, 2014). Several strategies have been used, including expression of soluble proteins in the cytoplasm, expression in the form of insoluble inclusion bodies, or export to the periplasm. The latter approach is favored for the production of proteins that contain disulfide bonds, because the periplasm is the only oxidizing compartment in wild‐type (WT) cells (Pooley, Merchante, & Karamata, 1996). We have shown that the Tat system can export hGH with high efficiency and that it is disulfide bonded (Alanen et al, 2015). We report that the system is able to produce purified, disulfide bonded, active hGH at levels that are calculated to be between 2 and 5 g/L culture

| MATERIALS AND METHODS

| RESULTS

Findings

| DISCUSSION