This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his,tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (lMAC). When using Co2+ 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 Cu2+ < Ni2+ < Zn2+ < Co2+ 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, in forming a tridentate with Me2+-IDA than in forming a bidentate with Me2+-NTA. The more flexible binding allows for multisite interactions over the protein. (C) 2000 John Wiley & Sons, Inc.