title: Cyanogen bromide-activated coupling: DNA catalytic chromatography purification of EcoRI endonuclease.
abstract: A method to purify enzymes utilizing their specific biological affinity and catalytic specificity is described. For this chromatographic technique, an enzyme binds immobilized substrate coupled to a column in the absence of a cofactor required for catalysis but permissive for substrate binding. After washing, the missing cofactor is added to the column mobile phase, and the enzyme converts substrate into product and elutes from the column. A single-step purification of EcoRI endonuclease using a sequence-specific DNA column (containing the GAATTC motif coupled to cyanogen bromide-activated Sepharose 4B) binds EcoRI in the absence of Mg2+ and elutes when Mg2+ is applied in a highly purified state. Although the method described is specific for EcoRI, it can be readily modified for the purification of DNA polymerases and other enzymes. Furthermore, many of the same materials are also used for transcription factor purification. This protocol can be completed within 4-6 d.
authors.affiliation: Department of Chemistry, University of Texas at San Antonio, One UTSA Circle, San Antonio, Texas 78249, USA.
authors.forename: Linda I
authors.initials: LI
authors.lastname: Nagore
authors.forename: Sucharita
authors.initials: S
authors.lastname: Mitra
authors.forename: Daifeng
authors.initials: D
authors.lastname: Jiang
authors.forename: Shoulei
authors.initials: S
authors.lastname: Jiang
authors.forename: YanWen
authors.initials: Y
authors.lastname: Zhou
authors.forename: Magda
authors.initials: M
authors.lastname: Loranc
authors.forename: Harry W
authors.initials: HW
authors.lastname: Jarrett
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D003488:Cyanogen Bromide / Q000737:chemistry; D015246:Deoxyribonuclease EcoRI / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D015246:Deoxyribonuclease EcoRI; D003488:Cyanogen Bromide