3 years ago

Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae

Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae
Balaji M. Rao, Karthik Tiruthani, Adam Mischler, Carlos A. Cruz-Teran
The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by “self-cleaving” 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence—with ∼50% efficiency of ribosomal skipping—between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein–Aga2 fusion that is tethered to the yeast cell surface. We show that binding proteins derived from the Sso7d scaffold and the homodimeric enzyme glucose oxidase can be simultaneously secreted solubly and expressed as yeast cell surface fusions using the F2A-based system. Furthermore, a combinatorial library of Sso7d mutants can be screened to isolate binders with higher affinity for a model target (lysozyme), and the pool of higher affinity binders can be characterized in soluble form. Significantly, we show that both N- and C-terminal fusions to Aga2 can be simultaneously secreted solubly and displayed on the cell surface; this is particularly advantageous because protein functionality can be affected by the specific position of Aga2 in the protein fusion. We expect that the F2A-based yeast surface display and secretion system will be a useful tool for protein engineering and enable efficient characterization of individual clones isolated from combinatorial libraries.

Publisher URL: http://dx.doi.org/10.1021/acssynbio.7b00144

DOI: 10.1021/acssynbio.7b00144

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