title: Suitability of immobilized metal affinity chromatography for protein purification from canola.
abstract: This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his(6)-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co(2+) with iminodiacetate (IDA) as the chelating ligand, beta-glucuronidase-his(6) (GUSH6) can be purified from canola protein extract with almost homogeneous purity in a single chromatographic step. The discrimination with which metal ions bound native canola proteins followed the order Cu(2+) < Ni(2+) < Zn(2+) < Co(2+) in regard to elimination of proteins coeluted with the fusion protein. IDA- and nitrilotriacetate (NTA)-immobilized metal ions showed different binding patterns, whose cause is attributed to a more rigid binding orientation of the his(6) in forming a tridentate with Me(2+)-IDA than in forming a bidentate with Me(2+)-NTA. The more flexible binding allows for multisite interactions over the protein.
authors.affiliation: Department of Chemical Engineering, 2114 Sweeney, Iowa State University, Ames, Iowa 50011, USA. cglatz@iastate.edu
authors.forename: C M
authors.initials: CM
authors.lastname: Zhang
authors.forename: S A
authors.initials: SA
authors.lastname: Reslewic
authors.forename: C E
authors.initials: CE
authors.lastname: Glatz
mesh_terms: D001937:Brassica / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D008670:Metals; D010940:Plant Proteins / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
chemical_list: D008670:Metals; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins