Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site.
The peptide sequence (N)DKTH(C) was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanized gamma1 Fab' as a model protein. Here we show that conservative mutations to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved. They were cleaved more efficiently by Cu(2+), such that cleavage reactions could be shorter, of lower pH or at a lower temperature. Some were even found to be measurably cleaved by Ni(2+). Use of these new cleavage sequences along with cupric ions may provide a more rapid and less harsh method for cost-effective, large-scale proteolytic cleavage of fusion proteins and peptides.
Humphreys
D P
DP
Celltech Therapeutics, 216 Bath Road, Slough, Berkshire SL1 4EN, UK. dhumphre@celltech.co.uk
King
L M
LM
West
S M
SM
Chapman
A P
AP
Sehdev
M
M
Redden
M W
MW
Glover
D J
DJ
Smith
B J
BJ
Stephens
P E
PE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003300:Copper / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D007140:Immunoglobulin Fab Fragments; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D003300:Copper; D009532:Nickel