A Novel Potent Carrier for Unconventional Protein Export in Ustilago maydis.

Magnus Philipp,Kira Müntjes,Kai P Hussnaetter,Michèle Reindl,Michael Feldbrügge,Kerstin Schipper

doi:10.3389/fcell.2021.816335

Magnus Philipp, Kira Müntjes + Show 4 more

Open Access

https://doi.org/10.3389/fcell.2021.816335

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Abstract

Recombinant proteins are ubiquitously applied in fields like research, pharma, diagnostics or the chemical industry. To provide the full range of useful proteins, novel expression hosts need to be established for proteins that are not sufficiently produced by the standard platform organisms. Unconventional secretion in the fungal model Ustilago maydis is an attractive novel option for export of heterologous proteins without N-glycosylation using chitinase Cts1 as a carrier. Recently, a novel factor essential for unconventional Cts1 secretion termed Jps1 was identified. Here, we show that Jps1 is unconventionally secreted using a fusion to bacterial β-glucuronidase as an established reporter. Interestingly, the experiment also demonstrates that the protein functions as an alternative carrier for heterologous proteins, showing about 2-fold higher reporter activity than the Cts1 fusion in the supernatant. In addition, Jps1-mediated secretion even allowed for efficient export of functional firefly luciferase as a novel secretion target which could not be achieved with Cts1. As an application for a relevant pharmaceutical target, export of functional bi-specific synthetic nanobodies directed against the SARS-CoV2 spike protein was demonstrated. The establishment of an alternative efficient carrier thus constitutes an excellent expansion of the existing secretion platform.

Highlights

The market for recombinant proteins like biopharmaceuticals is steadily increasing (Walsh 2018)
We applied the well-established β-glucuronidase (Gus) reporter system (Figure 1A,B). This bacterial enzyme is largely inactivated upon secretion through the eukaryotic endomembrane system. It is released in a functional state via unconventional secretion in yeast cells of U. maydis (Stock et al, 2012)
To assay unconventional secretion of Jps1, a strain expressing a Gus-Jps1 fusion protein was generated in the background of the octuple protease-deletion laboratory strain AB33P8Δ (Figure 1A) (Terfrüchte et al, 2018)

Summary

Introduction

The market for recombinant proteins like biopharmaceuticals is steadily increasing (Walsh 2018). Proteins are mostly targeted via the endomembrane system by N-terminal signal peptides for secretion (Viotti 2016). The term unconventional secretion describes protein export that does not occur via the classical endomembrane system including endoplasmic reticulum and Golgi apparatus (Nickel 2010). Various routes for such alternative secretion events exist, including direct transfer across the plasma membrane via transporters or self-sustained translocation or vesicular pathways where membrane vesicles are hitchhiked for export (Nickel 2010; Rabouille 2017)

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Conclusion