Enhancing the Thermostability and solubility of a single-domain catalytic antibody.

Abstract

Catalytic antibodies have the ability to bind to and degrade antigens, offering a significant potential for therapeutic use. The light chain of an antibody, UA15-L, can cleave the peptide bond of Helicobacter pylori urease, thus inhibiting the spread of the bacteria. However, the variable domain of UA15-L has a poor thermostability and solubility. In this study, we employed a combined computational and experimental approach to enhance the protein's stability and solubility properties. The protein unfolding hotspots were initially identified using molecular dynamics simulations. Following this, a disulfide bond was designed in an unfolding hotspot to stabilize the protein. Subsequently, protein solubility was enhanced with the assistance of computational methods by introducing polar or charged residues on the protein surface. The combination of multiple mutations resulted in UA15-L variable domain variants with improved thermostability, solubility, expression, and enhanced activity at elevated temperatures. These variants represent promising candidates for further engineering of catalytic activity and specificity.