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 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.

Metal assisted peptide bond hydrolysis: Chemistry, biotechnology and toxicological implications

“…Although successful in this particular case, this approach was not converted into a generally applicable procedure [140,141].…”

Major Advances in the Hydrolysis of Peptides and Proteins by Metal Ions and Complexes

“…By using an anchoring amino acid side chain to substitute for the Nterminal group, Zn II should be capable of promoting hydrolysis of internal Ser(Thr)-Xaa sequences in intact proteins. In the case of Ni II [49], Pd II [89,91,92,96], Pt II [96], and even Cu II [32,33,75,90,94,95,98], hydrolytic cleavage of Ser(Thr)-Xaa protein sequences has been reported in the literature.…”

“…Work with small peptides has led to the use of Cu II and Ni II ions to cleave intact proteins [32,33,49,90,95,130]. Horse heart myoglobin was hydrolyzed by CuCl 2 at Gln91- Fig.…”

“…In the case of Cu II , Co III , Ni II , Pd II , and Pt II , intact proteins have also been cleaved [32][33][34]36,49,50,74,75,82,86,[89][90][91][92][93][94][95][96][97][98]. Although hydrolysis is sometimes accomplished at ~pH 7.0 and 37 °C [34][35][36]41,[47][48][49][50][51][52], either extreme pH, and/or elevated temperatures are ocassionally employed [32,33,[63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][82][83]…”

Metal complexes as artificial proteases in proteomics: A palladium(II) complex cleaves various proteins in solutions containing detergents

“…Copper(II) ions have been shown to cleave several proteins, including the hinge region of human IgG antibodies [20]. Follow-up studies with Cu(II) ions showed specific hydrolysis of peptide bonds on the amino side of ser-his and thr-his sequences at high temperatures and basic conditions [21,22]. Myoglobin was also specifically cleaved by a series of Cu(II) reagents after Gln91 and Ala84 [23].…”