The Purification of Heme Peroxidases from Escherichia coli Inclusion Bodies: A Success Story Shown by the Example of Horseradish Peroxidase

In the following chapter a purification process for recombinant Horseradish peroxidase (HRP) produced in Escherichia coli is described. This enzyme is a secretory plant oxidoreductase belonging to the large peroxidase family III within the peroxidase-catalase superfamily of enzymes. It has high biotechnological significance, however, the isolation of the enzyme from its natural source, the horseradish root, has several shortcomings, which makes the development of a recombinant production strategy interesting. The presented protocol covers all process steps from isolation to the final chromatography step; the enzyme is solubilized from insoluble inclusion bodies, refolded and concentrated to yield a high purity enzyme preparation which is comparable to the commercially available plant-derived HRP. Moreover, we believe that this procedure can also be used to process other peroxidases of family II and III of the plant peroxidase superfamily, as they all share the same relevant features like disulfide bonds and a heme group.