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.

Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment

“…CNBr assay was done using previously described protocol by Nagore et al [ 50 ]. GAD65 promoter and control DNA fragments were amplified by PCR followed be gel elution for purity.…”