Metal-chelate affinity chromatography.
Recombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxyl terminus can be purified using a resin containing nickel ions (Ni(2+)) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique, known as metal-chelate affinity chromatography (MCAC), can readily be performed with either native or denatured protein. This unit discusses techniques for creating a fusion protein consisting of the protein of interest with a histidine tail attached. A procedure for expression of histidine-tail fusion proteins and their purification in native form by MCAC is described, and two alternate protocols describe purification of histidine-tail fusion proteins by MCAC under denaturing conditions and their renaturation by either dialysis or solid-phase renaturation. Support protocols are provided for analysis of the purified product and regeneration of the NTA resin. All of these protocols are easily adaptable to any protein expression system.
Petty
K J
KJ
University of Texas Southwestern Medical Center, Dallas, Texas, USA.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011489:Protein Denaturation; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012117:Resins, Synthetic / Q000737:chemistry
D011993:Recombinant Fusion Proteins; D012117:Resins, Synthetic; D006639:Histidine; D009532:Nickel; D009571:Nitrilotriacetic Acid