Recombinant production of horseradish peroxidase in E. coli and application in activated prodrug cancer therapy

Abstract

Recombinant production of the enzyme horseradish peroxidase in Escherichia coli and application of the enzyme in targeted cancer treatment The plant enzyme horseradish peroxidase (HRP) is a key component for various medical applications. Conjugated to antibodies and polymers, it is frequently used in medical diagnostics, biosensors and immunostainings. Furthermore, it was shown that a combination of HRP and the plant hormone indole-3-acetic acid (IAA) can be successfully used for targeted cancer treatment. Cancer is the second most frequent cause of death in Europe. Chemo- and radiation therapies, which are commonly used to fight cancer, cause unpleasant and painful side effects. A targeted enzyme/prodrug therapy allows a more specific treatment. However, HRP has not been employed for this purpose yet, since the currently available HRP preparation is isolated from plant as a mixture of heterogeneously glycosylated isoenzymes, which are hard to purify and are not reconcilable for the human body.In project P24861-B19, funded by the Austrian Science Fund (FWF), we successfully tackled different challenges in using recombinant HRP for medical purposes. Amongst other things, we developed efficient strategies to recombinantly produce single HRP isoenzymes in the yeast Pichia pastoris, we designed downstream strategies providing highly purified enzyme, we glycol-engineered both HRP and the yeast to allow the production of more homogeneously glycosylated enzyme and we tested recombinant HRP variants for their potential use in targeted cancer treatment in vitro. Since we were able to produce active and stable HRP variants with a reduced and more homogeneous glycosylation pattern, which showed activity with IAA on different cancer cell lines, we concluded that surface glycosylation was actually not a prerequisite for enzyme activity. Consequently, in this follow-up project we propose to:• express HRP and mutated HRP variants in Escherichia coli both 1) as soluble enzyme in the periplasm of E. coli allowing straight-forward screening of engineered HRP variants, and 2) as inclusion bodies (IBs) in the cytoplasm of E. coli in high amounts, which are then processed and refolded,• biochemically characterize the recombinantly produced, unglycosylated enzyme variants,• test them with IAA and paracetamol,• develop a strategy to efficiently conjugate the mutated HRP lead candidate with antibodies and lectins as recognition ligands for cell-specific delivery approaches and• evaluate the mutated HRP lead candidate and its conjugates on a panel of human cancer cell lines grown in 2D and 3D models in vitro to investigate their potential in targeted cancer treatment.