Wagstaff, J.L. and Howard, M.J. and Williamson, R.A. (2010) Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin alphavbeta6 binding peptides for NMR. Mol Biosyst, 6 (12). pp. 2380-5. ISSN 1742-2051 (Electronic)1742-2051 (Linking).
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.
|Additional information:||Wagstaff, Jane L Howard, Mark J Williamson, Richard A BB/D526461/1/Biotechnology and Biological Sciences Research Council/United Kingdom Research Support, Non-U.S. Gov't England Molecular bioSystems Mol Biosyst. 2010 Dec;6(12):2380-5. Epub 2010 Oct 18.|
|Uncontrolled keywords:||Amino Acid Sequence Antigens, Neoplasm/*metabolism Chromatography, High Pressure Liquid Chromatography, Reverse-Phase Electrophoresis, Polyacrylamide Gel Integrins/*metabolism Isotope Labeling/*methods Magnetic Resonance Spectroscopy Molecular Sequence Data Peptides/chemistry/*metabolism Recombinant Fusion Proteins/*biosynthesis Repetitive Sequences, Amino Acid Solubility Steroid Isomerases/*metabolism|
|Subjects:||Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group|
|Depositing User:||Mark Howard|
|Date Deposited:||16 Aug 2011 03:53|
|Last Modified:||06 Sep 2011 06:29|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/28015 (The current URI for this page, for reference purposes)|
- Depositors only (login required):