Thermal analysis of protein stability and ligand binding in complex media

Abstract

Screening of ligands that can bind to biologic products of in vitro expression systems typically requires some purification of the expressed biologic target. Such purification is often laborious and time consuming as well as a limiting challenge. What is required, representing an enormous advantage, is the ability to screen expressed proteins in the crude lysate stage without purification. For that purpose, we explore here, the utility of differential scanning calorimetry (DSC) measurements for detecting the presence of specific proteins and their interactions with ligands in the complex media where they were prepared, i.e. crude lysates. Model systems were designed to mimic analogous conditions comparable to those that may be encountered in actual in vitro expression systems. Results are reported for several examples where DSC measurements distinctly showed differences in the thermal denaturation behaviors of the crude lysate alone, proteins and proteins plus binding ligands added to the crude lysate. Results were obtained for Streptavidin/Biotin binding in E. coli lysate, and binding of Angiotensin Converting Enzyme 2 (ACE2) by captopril or lisinopril in the lysate supernatant derived from cultured Human Kidney cells (HEK293). ACE2 binding by the receptor binding domain (RBD) of SARS-CoV-2 was also examined. Binding of ACE2 by RBD and lisinopril were similar and consistent with the reported ACE2 inhibitory activity of lisinopril.