Bacterial Inner Membrane Display for Isolating Intracellular Antibodies from a Naïve Library

Antibodies that function intracellularly (intracellular antibodies) are promising tools for knocking down intracellular protein activity, but they present a protein engineering challenge because the reducing environment in the cytosol prevents formation of the disulfide bonds normally required for antibody folding and function. In our previous work, we developed an Escherichia coli inner membrane display platform that displays proteins that correctly fold in the E. coli cytoplasm by harnessing the intracellular folding quality control of the twin-arginine translocation pathway. Our present work used this platform to display a naïve library of single-chain variable fragment (scFv) antibodies with diversity in the third complementarity-determining regions (CDR3s) of the light and heavy chains. We then screened the library for binding to survivin, an apoptosis inhibitor that is upregulated in cancer cells. Our first round of screening isolated an scFv that binds to survivin and is expressed solubly in the E. coli cytoplasm. Using this isolate as the parent for random mutagenesis, we synthesized a second generation library and isolated additional scFvs that demonstrate improved binding to survivin and improved solubility compared to the intracellular scFv isolated in the first round of screening. Our results show the utility of the inner membrane display platform for isolating high-affinity, highly soluble intracellular antibodies that bind to clinically relevant targets.