Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin alphavbeta6 binding peptides for NMR

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Wagstaff, Jane L. ; Howard, Mark J. ; Williamson, Richard A. (2010)

The integrin alphavbeta6 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 alphavbeta6 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.
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