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 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.
Humphreys
D P
DP
Celltech Therapeutics, Slough, Berkshire, UK.
Smith
B J
BJ
King
L M
LM
West
S M
SM
Reeks
D G
DG
Stephens
P E
PE
D000596:Amino Acids / Q000737:chemistry; D002616:Chemical Engineering / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D016296:Mutagenesis; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids; D013696:Temperature; D013997:Time Factors
D000596:Amino Acids; D007140:Immunoglobulin Fab Fragments; D009842:Oligopeptides; D010455:Peptides; D003300:Copper; C085706:FLAG peptide