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Issue 12, 2010
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Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin αvβ6 binding peptides for NMR

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Abstract

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 15N and 13C 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.

Graphical abstract: Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin αvβ6 binding peptides for NMR

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Publication details

The article was received on 16 Jul 2010, accepted on 22 Sep 2010 and first published on 18 Oct 2010


Article type: Method
DOI: 10.1039/C0MB00105H
Mol. BioSyst., 2010,6, 2380-2385

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    Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin αvβ6 binding peptides for NMR

    J. L. Wagstaff, M. J. Howard and R. A. Williamson, Mol. BioSyst., 2010, 6, 2380
    DOI: 10.1039/C0MB00105H

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