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.

Immobilization of bacteriophage Qβ on metal-derivatized surfaces via polyvalent display of hexahistidine tags

“…Interestingly, the nature of the immobilized cation did not affect the elution profile of Qb-(His 6 ) 37 : HiTrap IMAC columns charged with any of Zn(II), Ni(II), Co(II), or Cu(II), gave rise to very similar elution profiles for Qb-(His 6 ) 37 , with peak elution occurring near 250 mM imidazole (Table S1). This insensitivity is unusual as other reports have generally described some metal-dependent retention of affinity-tagged proteins during IMAC [23][24][25].…”

Antibody Capture from Corn Endosperm Extracts by Packed Bed and Expanded Bed Adsorption

“…Thus, many of the opportunities to reduce the price of recombinant therapeutics derived from plants reside in the downstream portion of the process. To this end, a number of unit operations have been examined for the recovery of a recombinant protein from plants: plant tissue processing and protein extraction (12)(13)(14)(15)(16), precipitation (17,18), various modes of chromatography (19)(20)(21)(22), and expanded bed adsorption (23,24).…”

Copper complexed polymer carriers for IgG adsorption

“…IMAC helps in the separation of materials by their selective interaction, on the basis of their affinities, with metal ions. In the case of proteins, the separation is based on their differential binding abilities with chelated metal ions in a support [18][19][20][21][22] and this is due to the presence of histidine side chains which is a major factor contributing to the overall affinity of proteins for immobilized metal ions. The other factors contributing to bimolecular adsorption are aromatic amino acids and amino terminus of the peptides, microenvironment of the binding residue, co-operation between neighbouring amino acid side chains and local conformations [23].…”