Suitability of immobilized metal affinity chromatography for protein purification from canola

✍ Scribed by Chen-Ming Zhang; Susan A. Reslewic; Charles E. Glatz

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 212 KB
• Volume: 68
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/(sici)1097-0290(20000405)68:1<52::aid-bit6>3.0.co;2-a

✦ Synopsis

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.