posted on 2024-03-14, 15:22authored byNicola
M. O’Riordan, Vanja Jurić, Sarah K. O’Neill, Aoife P. Roche, Paul W. Young
Saccharomyces cerevisiae is an attractive
host for the expression of secreted proteins in a biotechnology context.
Unfortunately, many heterologous proteins fail to enter, or efficiently
progress through, the secretory pathway, resulting in poor yields.
Similarly, yeast surface display has become a widely used technique
in protein engineering but achieving sufficient levels of surface
expression of recombinant proteins is often challenging. Signal peptides
(SPs) and translational fusion partners (TFPs) can be used to direct
heterologous proteins through the yeast secretory pathway, however,
selection of the optimal secretion promoting sequence is largely a
process of trial and error. The yeast modular cloning (MoClo) toolkit
utilizes type IIS restriction enzymes to facilitate an efficient assembly
of expression vectors from standardized parts. We have expanded this
toolkit to enable the efficient incorporation of a panel of 16 well-characterized
SPs and TFPs and five surface display anchor proteins into S. cerevisiae expression cassettes. The secretion
promoting signals are validated by using five different proteins of
interest. Comparison of intracellular and secreted protein levels
reveals the optimal secretion promoting sequence for each individual
protein. Large, protein of interest-specific variations in secretion
efficiency are observed. SP sequences are also used with the five
surface display anchors, and the combination of SP and anchor protein
proves critical for efficient surface display. These observations
highlight the value of the described panel of MoClo compatible parts
to allow facile screening of SPs and TFPs and anchor proteins for
optimal secretion and/or surface display of a given protein of interest
in S. cerevisiae.