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

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.

An Engineered Pathway for Production of Terminally Sialylated N-glycoproteins in the Periplasm of Escherichia coli

“…A favored expression strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation (Karyolaimos et al, 2019;Tripathi and Shrivastava, 2019). Yields of several g/L of active human growth hormone, containing two disulfide bonds and exported to the periplasm using the Tat pathway, were recently demonstrated in E. coli "TatExpress" strains (Guerrero et al, 2019). The lack of a natural pathway to achieve N-glycosylation remains a major limitation of E. coli as an expression host, however.…”

Engineering of the Bacillus subtilis PhoD signal peptide to direct high-level, Tat-specific export of a single-chain antibody fragment to the periplasm in Eschericha coli

“…[13]). We therefore tested the ability of other Tat signal peptides to direct high-e ciency export of biopharmaceuticals, and used a model biopharmaceutical that has shown to be exported by Tat in previous studies: a single chain variable fragment (scFv) [14]. We used three E. coli Tat signal peptides, taken from MdoD, AmiC and YcbK, and the Tat signal peptide from Bacillus subtilis PhoD, which is transported by the TatAdCd system in B. subtilis (reviewed in [16]).…”

“…Recent studies have shown that the Tat pathway can export human growth hormone at levels of several g/L in fed-batch fermentation cultures of E. coli 'TatExpress' strains that simultaneously over-express the Tat machinery [14]. Clearly, this platform is capable of high-level protein secretion to the periplasm, but previous work has already pointed to potential limitations in the current platform.…”

“…Most of the protein is thus lost during puri cation, which makes quanti cation di cult. However, the scFv has the same His tag as that present on hGH which which was exported at levels of 2-5 g/L culture in a previous study [14], and we have therefore used semi-quantitiative immunoblotting with the scFv and hGH run on the same gels and blots. This indicates that the levels of scFv exported with the PhoDn signal peptide reach approximately 1g protein per litre of culture, while the TorA-scFv is exported at signi cantly lower levels.…”

“…The system is able to export a wide range of heterologous proteins if a Tat-speci c signal peptide is attached; examples include green uorescent protein [12], alkaline phosphatase [7] and a series of biopharmaceuticals such as human growth hormone (hGH) and antibody single chain variable fragments [13]. Until recently it was unclear whether the Tat system could handle the large export rates required for industrial applications, but we recently showed that hGH could be exported at very high rates (several grams of protein per litre in fed-batch fermentation) in E. coli 'TatExpress' strains that over-express the tatABC operon to cope with high-level export [14].…”