New approaches for functional expression of recombinant horseradish peroxidase C in Escherichia coli

Horseradish peroxidase (HRP) is an important enzyme in bio- and immunochemical analysis. In a first approach we expressed HRP with a C-terminal histidine tag in the Escherichia coli (E. coli) periplasm. On the one hand this led to functionally active HRP-His at a low yield of 0.5 mg/l of culture medium. On the other hand the affinity tag introduced allowed for optimization of the downstream processing of HRP-His refolded from inclusion bodies, thereby increasing the yield of homogeneous enzyme to 8-10 mg/l of E. coli culture medium when expressed conventionally in E. coli cytosol as the second approach. The final refolding/reconstitution protocol includes crucial gel filtration steps to remove constituents of the refolding medium and, in particular, imidazole from the active site of HRP-His, resulting in spectral (Soret band maximum of 403 nm) and catalytic properties of the refolded HRP-His (1160 U/mg with 2,2'-Azino-bis[3-ethylbenzthiazoline-6-sulfonate] as substrate) indistinguishable from those of the plant-derived HRP.