title: A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli.
abstract: Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E. coli and investigated and compared both strategies, (a) the production of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the production of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titre of 100mgL-1cultivation broth, and were able to produce 48mg active HRP per litre cultivation broth in the periplasm. In terms of biochemical properties, soluble HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and production. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amounts, which are then refolded.
authors.affiliation: TU Wien, Institute of Chemical, Environmental and Biological Engineering, Research Area Biochemical Engineering, Gumpendorfer Strasse 1a, 1060 Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gundinger
authors.affiliation: TU Wien, Institute of Chemical, Environmental and Biological Engineering, Research Area Biochemical Engineering, Gumpendorfer Strasse 1a, 1060 Vienna, Austria. Electronic address: oliver.spadiut@tuwien.ac.at.
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
mesh_terms: D019149:Bioreactors; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D006735:Horseradish Peroxidase
keywords: Escherichia coli; Horseradish peroxidase; Inclusion body; Periplasm; Refolding