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Protein Engineering, Vol. 12, No. 2, 179-184, February 1999
© 1999 Oxford University Press

Efficient site specific removal of a C-terminal FLAG fusion from a Fab' using copper(II) ion catalysed protein cleavage

David P. Humphreys1, Bryan J. Smith, Lloyd M. King, Shauna M. West, Dominic G. Reeks and Paul E. Stephens

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

The peptide sequence NDKTHC was investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanised {gamma}1 Fab' as a model protein. The native upper hinge NDKTHC sequence was mutated to create a site resistant to cleavage by cupric ions and a NDKTHC sequence introduced between the hinge and a C-terminal FLAG peptide. Incubation of Fab' with Cu2+ at 62°C at alkaline pHs resulted in removal of the FLAG peptide with efficiencies of up to 86%. Cleavage conditions did not detrimentally affect the Fab' protein. Use of the NDKTHC sequence along with cupric ions may provide a cost-effective method for large scale proteolytic cleavage of fusion proteins.

Keywords: copper (II)/cupric/Fab'/FLAG peptide/protein cleavage

1 To whom correspondence should be addressed


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Home page
 Protein Eng Des SelHome page
D. P. Humphreys, L. M. King, S. M. West, A. P. Chapman, M. Sehdev, M. W. Redden, D. J. Glover, B. J. Smith, and P. E. Stephens
Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site
Protein Eng. Des. Sel., March 1, 2000; 13(3): 201 - 206.
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