Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Grigorenko, V; Chubar, T; Kapeliuch, Y; Borchers, T; Spener, F; Egorov, A.
New approaches for functional expression of recombinant horseradish peroxidase C in Escherichia coli
BIOCATAL BIOTRANSFORM. 1999; 17(5): 359-379.
Doi: 10.3109/10242429909015236
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- Co-Autor*innen der Med Uni Graz
- Spener Friedrich
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- Abstract:
- 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.
- Find related publications in this database (Keywords)
- horseradish peroxidase
- recombinant holoprotein
- E-coli
- periplasm
- refolding