Purification and Crystallization of Chloromuconolactone Dehalogenase ClcF from Rhodococcus opacus 1CP

Abstract

The protocol describes the generation of variants of chloromuconolactone dehalogenase from Rhodococcus opacus (R. opacus) 1CP. ClcF is a multimeric protein, which catalyses the dechlorination of 5-chloromuconolactone to cis-dienelactone in the 3-chlorocatecholic acid degradation pathway. The protocol describes the workflow for the purification and subsequent crystallization of the enzyme. The used workflow and the described techniques could be easily adapted to any other protein/enzyme intended to be crystallized by the potential user for subsequent structure determination. The protocol does not involve expensive specialized equipment which allows the use in standard laboratories not specially dedicated to macromolecular crystallography.