His6 tag-assisted chemical protein synthesis.
To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses.
Bang
Duhee
D
Institute for Biophysical Dynamics and Department of Chemistry, University of Chicago, Chicago, IL 60637, USA.
Kent
Stephen B H
SB
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006639:Histidine / Q000737:chemistry; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D010940:Plant Proteins / Q000138:chemical synthesis / Q000737:chemistry; D017510:Protein Folding; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D020449:Repetitive Sequences, Amino Acid; D013816:Thermodynamics
D008024:Ligands; D009842:Oligopeptides; D010940:Plant Proteins; D011506:Proteins; D006639:Histidine; C024934:crambin protein, Crambe abyssinica