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.
Xiao
Junpeng
J
Department of Chemistry and Interdisciplinary Biochemistry Graduate Program, Indiana University,Bloomington, Indiana 47405, USA.
Burn
Alex
A
Tolbert
Thomas J
TJ
D001622:Betaine / Q000494:pharmacology*; D001665:Binding Sites; D000077560:Enfuvirtide; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility / Q000187:drug effects; D013379:Substrate Specificity; D019820:Xanthine / Q000378:metabolism
D015700:HIV Envelope Protein gp41; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D000077560:Enfuvirtide; D019820:Xanthine; D001622:Betaine; D007041:Hypoxanthine Phosphoribosyltransferase