Rational design approach to improve the solubility of the β-sandwich domain 1 of a thermophilic protein.

The β-sandwich domain 1 (SD1) of islandisin is a stable thermophilic protein with surface loops that can be redesigned for specific target binding, architecturally comparable to the variable domain of immunoglobulin (IgG). SD1's propensity to aggregate due to incorrect folding and subsequent accumulation in Escherichia coli inclusion bodies limits its use in biotechnological applications. We rationally designed SD1 for improved variants that were expressed in soluble forms in E. coli while maintaining the intrinsic thermal stability of the protein (melting temperature (Tm) = 73). We used FoldX's ΔΔG predictions to find beneficial mutations and aggregation-prone regions (APRs) using Tango. The S26K substitution within protein core residues did not affect protein stability. Among the soluble mutants studied, the S26K/Q91P combination significantly improved the expression and solubility of SD1. We also examined the effects of the surface residue, pH, and concentration on the solubility of SD1. We showed that the surface polarity of proteins had little or no effect on solubility, whereas surface charges played a substantial role. The storage stability of several SD1 variants was impaired at pH values near their isoelectric point, and pH levels resulting in highly charged groups. We observed that mutations that create an uneven distribution of charged groups on the SD1 surface could enhance protein solubility by eliminating favorable protein-protein surface charge interactions. Our findings suggest that SD1 is mutationally tolerant to new functionalities, thus providing a novel perspective for the application of rational design to improve the solubility of targeted proteins.