Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans.
The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable and prohibitively expensive when the peptides are large (e.g., >30 amino acids) or when isotope labeling is required for NMR studies. Expression and purification of large quantities of unfolded peptides in E. coli have also proved to be difficult even when the desired peptides are carried by fusion proteins such as GST. We have developed a peptide expression system that utilizes a novel fusion protein (SFC120) which is highly expressed and directs the peptides to inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields of peptide expression. The expressed peptides can be liberated from the carrier protein by CNBr cleavage at engineered methionine sites or through proteolysis by specific proteases for peptides containing methionine residues. In the present systems, we use CNBr, due to the absence of methionine residues in the target peptides, although other cleavage sites can be easily inserted. We report the production of six unfolded protein fragments of different composition and lengths (19 to 48 residues) derived from the virulent effector kinases, Cla4 and Ste20 of Candida albicans. All six peptides were produced with high yields of purified material (30-40 mg/l in LB, 15-20 mg/l in M9 medium), pointing to the general applicability of this expression system for peptide production. The enrichment of these peptides with (15)N, (15)N/(13)C and even (15)N/(13)C/(2)H isotopes is presented allowing speedy assignment of poorly-resolved resonances of flexible peptides.
Osborne
Michael J
MJ
Biomolecular NMR and Protein Research Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2, Canada.
Su
Zhengding
Z
Sridaran
Vasanth
V
Ni
Feng
F
D000595:Amino Acid Sequence; D001665:Binding Sites; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D016384:Consensus Sequence; D003488:Cyanogen Bromide / Q000494:pharmacology; D003903:Deuterium; D004926:Escherichia coli / Q000378:metabolism; D002479:Inclusion Bodies; D047908:Intracellular Signaling Peptides and Proteins; D007553:Isotope Labeling / Q000379:methods*; D020930:MAP Kinase Kinase Kinases; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D015202:Protein Engineering; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D029701:Saccharomyces cerevisiae Proteins; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D002247:Carbon Isotopes; D047908:Intracellular Signaling Peptides and Proteins; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D008715:Methionine; D003903:Deuterium; C095212:CLA4 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D003488:Cyanogen Bromide