Structural Characterization of Nanoyeast Single-Chain Fragment Variable Affinity Reagents

Abstract

Nanoyeast single-chain variable fragments (nanoyeast-scFv) are a new class of low-cost and stable protein capture agents developed as alternatives to full length monoclonal antibodies for use in immunosensors. Physical characteristics that impart nanoyeast-scFv with these advantages have yet to be investigated. We investigate the structure, size and surface loading of nanoyeast-scFv to better understand its ability to specifically and sensitively capture proteins of interest while retaining activity in solution. Nanoyeast-scFv fragments were found to be globular in structure and heterogeneous in size, typically <100 nm, and were found to aggregate in solution. Optimal fragment size was determined to be <220 nm, with larger fragments interfering with the electrochemical biosensor. We hypothesize that the stability of nanoyeast-scFv is conferred in part by components of the yeast cell wall fragments, which provide stability to the associated scFv both during selection, lyophilization, and rehydration.