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

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.