Chen-Ming Zhang, Susan A. Reslewic, Charles E. Glatz
This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his6-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co2+ with iminodiacetate (IDA) as the chelating ligand, β-glucuronidase-his6 (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 his6 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. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 52–58, 2000.