title: Purification of recombinant human carbonic anhydrase-II by metal affinity chromatography without incorporating histidine tags.
abstract: Due to their involvement in diverse pathological conditions, carbonic anhydrases have been the targets of drug developments for the treatments of glaucoma, epilepsy, high altitude sickness, as well as cancer. Of about 14 isozymes of carbonic anhydrases, carbonic anhydrase-II (hCA-II) has been most extensively investigated from the structural, functional, and inhibitor design point of view. We discovered that hCA-II preferentially binds to the Sepharose-iminodiacetate (IDA)-Zn(2+) column, and such binding does not require incorporation of either N- or C-terminal histidine tags in the protein structure. By using the Sepharose-IDA-Zn(2+) affinity column, we purified the Escherichia coli expressed hCA-II with an overall recovery of 76%. The purified enzyme showed a single band on the SDS-PAGE. Due to ease in preparing the Sepharose-IDA-Zn(2+) column, and purifying hCA-II just in one step, the overall protocol will be ideal for producing bulk quantities of the enzyme for high throughput screening of inhibitors.
authors.affiliation: Department of Chemistry and Molecular Biology, North Dakota State University, Fargo, ND 58105, USA.
authors.forename: Abir L
authors.initials: AL
authors.lastname: Banerjee
authors.forename: Michael
authors.initials: M
authors.lastname: Swanson
authors.forename: Sanku
authors.initials: S
authors.lastname: Mallik
authors.forename: D K
authors.initials: DK
authors.lastname: Srivastava
mesh_terms: D001665:Binding Sites; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000302:isolation & purification*; D002845:Chromatography; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007098:Imino Acids / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry
chemical_list: D007098:Imino Acids; D011994:Recombinant Proteins; C030699:copper(II)-iminodiacetate; D006639:Histidine; D012685:Sepharose; D024402:Carbonic Anhydrase II