Enhancing the crystallisation of insulin using amino acids as soft-templates to control nucleation

Abstract

Amino acids have been widely used in protein formulations to increase the protein's stability. In this study, amino acids have been introduced as soft-templates to control the nucleation of proteins. L-Arginine, L-glycine or L-leucine with concentrations between 0.01 M and 0.1 M were used in a hanging drop vapor diffusion set-up to investigate their role in the crystallisation of human insulin as a model protein at low supersaturation. All amino acids were in a dissolved state. Here we show that L-arginine and L-leucine clearly enhance the nucleation significantly. On the other hand, L-glycine does not enhance nucleation of human insulin at low supersaturation. We hypothesize that it is the intermolecular interaction between the protein's residues and the amino acid's residues that results in an enhancement in the formation of the initial protein nuclei. To prove that the enhanced nucleation is of a kinetic nature, the solubility of insulin in amino acid rich solution was also investigated. The solubility results show that amino acids increase insulin solubility. From that we derive that the enhancement in nucleation is not due to a change in the thermodynamic equilibrium between the crystalline and bulk-liquid phase. As this approach of using amino acids to enhance nucleation is based on a dissolved state in solution, we introduce here the concept of protein crystallisation by soft-templating.