Disulfide-compatible phage-assisted continuous evolution in the periplasmic space.

The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution.