Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin αvβ6 binding peptides for NMR.
The integrin αvβ6 is up-regulated in several cancers and has clinical potential for both tumour imaging and therapy. Peptide ligands have been developed which show good binding specificity for αvβ6 and provide an opportunity to study the interaction in more detail by NMR. Such studies ideally require (15)N and (13)C labelled peptides, and recombinant expression within E. coli provides a cost effective way of generating isotopically labelled proteins and peptides. In this study we have used an insoluble fusion partner (ketosteroid isomerase) to produce high yields of recombinant peptide. The insoluble nature of the fusion allowed simple product recovery by cell lysis and centrifugation, and thorough washing of the insoluble pellet to remove contaminating proteins avoided the need for nickel-affinity chromatography in denaturing conditions which is the standard procedure. The protocol described here is convenient to scale-up and requires only one chromatography step (reverse-phase HPLC) which is comparable to solid-phase synthesis.
Protein Science Group, School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK.
Jane L
JL
Wagstaff
Mark J
MJ
Howard
Richard A
RA
Williamson
D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D016023:Integrins / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D020449:Repetitive Sequences, Amino Acid; D012995:Solubility; D013251:Steroid Isomerases / Q000378:metabolism*
D000951:Antigens, Neoplasm; D016023:Integrins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C073878:integrin alphavbeta6; D013251:Steroid Isomerases