Increasing solubility of proteins and peptides by site-specific modification with betaine

Proteins and peptides with low solubility and which aggregate are often encountered in biochemical studies and in pharmaceutical applications of polypeptides. Here, we report a new strategy to improve solubility and prevent aggregation of polypeptides using site-specific modification with the small molecule betaine, which contains a quaternary ammonium moiety. Betaine was site-selectively attached to the N-termini of two aggregation-prone polypeptide models, the bacterial enzyme xanthine-guanine phosphoribosyltransferase (CG-GPRT) and the HIV entry inhibitor peptide CG-T20, utilizing native chemical ligation. N-terminal cysteines for the betaine ligation reactions were generated from His-tagged fusion proteins using TEV protease cleavage. Ligation of the betaine thioester (1) to the N-terminal cysteine-containing polypeptide models proceeded in high yield, though denaturing conditions were required for CG-T20 due to the hydrophobic nature of this peptide. CD spectroscopy and GPRT activity assays indicate that the betaine modification of CG-GPRT and CG-T20 does not significantly affect structure or activity of the polypeptides. Solubility and turbidity measurements of betaine-modified and unmodified polypeptides demonstrate that betaine modification can greatly increase solubility. Finally, it is shown that betaine-modified CG-T20 acts as an inhibitor of the aggregation of unmodified CG-T20.