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Suitability of immobilized metal affinity chromatography for protein purification from canola
Author(s) -
Zhang ChenMing,
Reslewic Susan A.,
Glatz Charles E.
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(20000405)68:1<52::aid-bit6>3.0.co;2-a
Subject(s) - chemistry , affinity chromatography , chelation , ligand (biochemistry) , canola , chromatography , metal , metal ions in aqueous solution , denticity , protein purification , ion chromatography , biochemistry , inorganic chemistry , organic chemistry , enzyme , receptor , food science
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, β‐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. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 52–58, 2000.