Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC)

Abstract

Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography.

DMSO tolerant NAD(P)H recycler enzyme from a pathogenic bacterium, Burkholderia dolosa PC543: effect of N‐/C‐terminal His Tag extension on protein solubility and activity

“…However, the decision regarding the relative positioning of the affinity tags remains difficult and depends on the primary structure and conformation of the protein [14]. A polyhistidine-tag is an affinity tag comprising an amino acid motif made up of at least six consecutive histidine residues usually located at the N-or C-terminus of a protein [19,20]. A polyhistidine-tag is an affinity tag comprising an amino acid motif made up of at least six consecutive histidine residues usually located at the N-or C-terminus of a protein [19,20].…”

“…In addition, the fusion of affinity tags has some perceived limitations, which are: misfolding and/or loss of activity or solubility of the protein, inability to use such proteins for X-ray crystallography or other physical characterization studies [15][16][17][18]. A polyhistidine-tag is an affinity tag comprising an amino acid motif made up of at least six consecutive histidine residues usually located at the N-or C-terminus of a protein [19,20]. Although His-tag engineering has been extensively carried out for protein purification, the effect of His-tagging on different terminal regions of enzymes has not been well considered.…”