A General Method to Screen Nanobodies for Cytochrome P450 Enzymes from a Yeast Surface Display Library

Abstract

The availability of yeast surface display nanobody (Nb) libraries offers a convenient way to acquire antigen-specific nanobodies that may be useful for protein structure–function studies and/or therapeutic applications, complementary to the conventional method of acquiring nanobodies through immunization in camelids. In this study, we developed a general approach to select nanobodies for cytochrome P450 enzymes from a highly diverse yeast display library. We tested our method on three P450 enzymes including CYP102A1, neuronal nitric oxide synthase (nNOS), and the complex of CYP2B4:POR, using a novel streamlined approach where biotinylated P450s were bound to fluorescent-labeled streptavidin for Nb screening. The Nb–antigen binders were selectively enriched using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). After two rounds of MACS, the population of positive binders was enriched by >5-fold compared to the naïve library. The subsequent FACS selection, with a gating of 0.1%, identified 634, 270, and 215 positive binders for CYP102A1, nNOS, and CYP2B4:POR, respectively. The positive binders for CYP102A1 were further triaged based on EC50 determined at various antigen concentrations. DNA sequencing of the top 30 binders of CYP102A1 resulted in 26 unique clones, 8 of which were selected for over-expression and characterization. They were found to inhibit CYP102A1-catalyzed oxidation of omeprazole with IC50 values in the range of 0.16–2.8 µM. These results validate our approach and may be applied to other protein targets for the effective selection of specific nanobodies.

Keywords

• P450
• nanobody
• biotinylation
• CYP102A1
• yeast display library