title: Application of Ni(II)-assisted peptide bond hydrolysis to non-enzymatic affinity tag removal.
abstract: In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His(6). This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Edyta
authors.initials: E
authors.lastname: Kopera
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Belczyk-Ciesielska
authors.forename: Wojciech
authors.initials: W
authors.lastname: Bal
mesh_terms: D000345:Affinity Labels / Q000737:chemistry / Q000302:isolation & purification*; D020816:Amino Acid Motifs; D006801:Humans; D006868:Hydrolysis; D019811:Hydroxylamine / Q000737:chemistry; D009532:Nickel / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D059748:Proteolysis; D012996:Solutions; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D000345:Affinity Labels; D009842:Oligopeptides; D012996:Solutions; D025801:Ubiquitin; D019811:Hydroxylamine; D009532:Nickel