Engineering and Adapting Disulfide-Containing Proteins to Enable Intracellular Functionality.

Disulfide bonds are key structural components ensuring proper protein folding. Those bonds, however, do not form in the reducing intracellular environment, often resulting in misfolded, unstable proteins with subpar function. To enable intracellular functionality of such proteins, disulfide-free variants with restored stability and function should be engineered. Here, we present a method to substitute the cysteine residues with optimal amino acid combinations using site saturation mutagenesis and screening for improved disulfide-free variants using yeast surface display. We provide a detailed protocol to directly screen for thermal stability of proteins using conformationally specific ligands, and an alternative to harness the correlation between protein stability and expression levels in yeast. We also discuss different strategies for randomizing cysteine residues and approaches for handling proteins with multiple disulfide bonds.