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

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 ggr;1 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 Cu 2+ 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 N DKTH C 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 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Protein Engineering, Design and Selection Oxford University Press

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

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 ggr;1 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 Cu 2+ 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 N DKTH C 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
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