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. 1999 Feb;12(2):179-84.
doi: 10.1093/protein/12.2.179.

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

D P Humphreys  1 , B J Smith, L M King, S M West, D G Reeks, P E Stephens
Affiliations

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

D P Humphreys et al. Protein Eng. 1999 Feb.

Abstract

The peptide sequence (N)DKTH(C) was investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanised gamma1 Fab' as a model protein. The native upper hinge (N)DKTH(C) sequence was mutated to create a site resistant to cleavage by cupric ions and a (N)DKTH(C) sequence introduced between the hinge and a C-terminal FLAG peptide. Incubation of Fab' with Cu2+ at 62 degrees 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 (N)DKTH(C) sequence along with cupric ions may provide a cost-effective method for large scale proteolytic cleavage of fusion proteins.

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