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1 Canadian Institutes of Health Research Group in Protein Structure and Function and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Reprint requests to: Brian D. Sykes, 4-19 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7; e-mail: Brian.Sykes{at}ualberta.ca; fax: (780) 492-0886.
Fusion protein constructs of the 56 amino acid globular protein GB-1 with various peptide sequences, coupled with the incorporation of a histidine tag for affinity purification, have generated high-yield fusion protein constructs. Methionine residues were inserted into the constructs to generate pure peptides following CNBr cleavage, yielding a system that is efficient and cost effective for isotopic labeling of peptides for NMR studies and other disciplines such as mass spectroscopy. Six peptides of varying sequences and hydrophobicities were expressed using this GB-1 fusion protein technique and produced soluble fusion protein constructs in all cases. The ability to easily express and purify recombinant peptides in high yields is applicable for biomedical research and has medicinal and pharmaceutical applications.
Keywords: NMR; GB-1; isotopic labeling; CNBr cleavage; affinity purification
Abbreviations: NMR, nuclear magnetic resonance • GB-1, B1 immunoglobulin domain of streptococcal protein G • cTnI, human cardiac troponin I • cTnT, human cardiac troponin T • CNBr, cyanogen bromide • His, 6-poly histidine • IPTG, isopropyl ß-D-thiogalactopyranoside • MW, molecular weight • NHE1, human sodium proton exchanger isoform 1 • CapZ
1, actin capping protein • 2xYT, 2X-yeast-tryptone media • HPLC, high performance liquid chromatography • MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight • HSQC, heteronuclear single quantum coherence • OD, optical density • EDTA, ethylenediaminetetraacetic acid • DSS, sodium 2,2-dimethyl-,2-silapantane-5-sulfonate
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