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Protein Engineering, Vol. 13, No. 3, 201-206, March 2000
© 2000 Oxford University Press

Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site

David P. Humphreys1, Lloyd M. King, Shauna M. West, Andrew P. Chapman, Mukesh Sehdev, Matthew W. Redden, David J. Glover, Bryan J. Smith and Paul E. Stephens

Celltech Therapeutics, 216 Bath Road, Slough, Berkshire SL1 4EN, UK

The peptide sequence NDKTHC was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanized {gamma}1 Fab' as a model protein. Here we show that conservative mutations to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved. They were cleaved more efficiently by Cu2+, such that cleavage reactions could be shorter, of lower pH or at a lower temperature. Some were even found to be measurably cleaved by Ni2+. Use of these new cleavage sequences along with cupric ions may provide a more rapid and less harsh method for cost-effective, large-scale proteolytic cleavage of fusion proteins and peptides.


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