Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans.

摘要: The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable prohibitively expensive when the are large (e.g., >30 amino acids) or isotope labeling required for NMR studies. Expression purification quantities unfolded in E. coli have also proved to be difficult even desired carried by fusion proteins as GST. We developed a peptide expression system that utilizes novel (SFC120) which highly expressed directs inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields expression. can liberated from carrier CNBr cleavage at engineered methionine sites through specific proteases containing residues. In present systems, we use CNBr, due absence residues target peptides, although other easily inserted. report six different composition lengths (19 48 residues) derived virulent effector kinases, Cla4 Ste20 Candida albicans. All were produced with purified material (30-40 mg/l LB, 15-20 M9 medium), pointing general applicability this production. enrichment these (15)N, (15)N/(13)C (15)N/(13)C/(2)H isotopes presented allowing speedy assignment poorly-resolved resonances flexible peptides.