Proteome-Scale Screening to Identify High-Expression Signal Peptides with Minimal N-Terminus Biases via Yeast Display

Abstract

Signal peptides are critical for the efficient expression and routing of extracellular and secreted proteins. Most protein production and screening technologies rely upon a relatively small set of signal peptides. Despite their central role in biotechnology, there are limited studies comprehensively examining the interplay between signal peptides and expressed protein sequences. Here, we describe a high-throughput method to screen novel signal peptides that maintain a high degree of surface expression across a range of protein scaffolds with highly variable N-termini. We find that the canonical signal peptide used in yeast surface display, derived from Aga2p, fails to achieve high surface expression for 42.5% of constructs containing diverse N-termini. To circumvent this, we have identified two novel signal peptides derived from endogenous yeast proteins, SRL1 and KISH, which are highly tolerant to diverse N-terminal sequences. This pipeline can be used to expand our understanding of signal peptide function, identify improved signal peptides for protein expression, and refine the computational tools used for signal peptide prediction.

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae

“…cerevisiae SPs and identified six that promoted high levels of secretion of an exogenous β-galactosidase enzyme. Holec et al conducted a proteome-scale screen to identify SPs that gave a consistent, high-level surface display of proteins with diverse amino-terminal sequences. Other studies have optimized the widely used α-factor mating pheromone pre-pro-SP through a variety of strategies to achieve enhanced secretion of diverse proteins of interest. − Bae and co-workers conducted a genome-wide screen to identify sequences termed translational fusion partners (TFPs) that enhanced the expression and/or secretion of several difficult-to-produce proteins.…”

“…Mori et al 7 screened a library of 60 S. cerevisiae SPs and identified six that promoted high levels of secretion of an exogenous β-galactosidase enzyme. Holec et al 8 conducted a proteome-scale screen to identify SPs that gave a consistent, high-level surface display of proteins with diverse amino-terminal sequences. Other studies have optimized the widely used α-factor mating pheromone pre-pro-SP through a variety of strategies to achieve enhanced secretion of diverse proteins of interest.…”

A yeast modular cloning (MoClo) toolkit expansion for optimization of heterologous protein secretion and surface display inSaccharomyces cerevisiae

“…Holec et al . (2022) (8) conducted a proteome-scale screen to identify SPs that gave consistent, high-level surface display of proteins with diverse amino-terminal sequences. Other studies have optimized the widely used α-factor mating pheromone pre-pro-SP through a variety of strategies to achieve enhanced secretion of diverse proteins of interest (9-12) .…”

“…Mori et al (2015) (7) screened a library of 60 S. cerevisiae SPs and identified six that promoted high levels of secretion of an exogenous β-galactosidase enzyme. Holec et al (2022) (8) conducted a proteome-scale screen to identify SPs that gave consistent, high-level surface display of proteins with diverse amino-terminal sequences. Other studies have optimized the widely used α-factor mating pheromone pre-pro-SP through a variety of strategies to achieve enhanced secretion of diverse proteins of interest (9)(10)(11)(12).…”

A new mRNA structure prediction based approach to identifying improved signal peptides for bone morphogenetic protein 2

“…The Signal Peptide Secretion Efficiency Database is a promising alternative approach that attempts to address the issue by providing a repository of results for SP, gene and host combinations, though currently only contains data for a small number of proteins in prokaryotes [29]. Recent advances in in vitro screening methods show promise, though require large and complex screens that are not feasible for many researchers [30,31]. There is still an unmet need for improved low-cost methods of choosing SP candidates.…”