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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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