Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study
The result's identifiers
Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11110%2F21%3A10441270" target="_blank" >RIV/00216208:11110/21:10441270 - isvavai.cz</a>
Result on the web
<a href="https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=40stTrjOMX" target="_blank" >https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=40stTrjOMX</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1371/journal.pone.0259837" target="_blank" >10.1371/journal.pone.0259837</a>
Alternative languages
Result language
angličtina
Original language name
Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study
Original language description
Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
30202 - Endocrinology and metabolism (including diabetes, hormones)
Result continuities
Project
<a href="/en/project/NV17-32727A" target="_blank" >NV17-32727A: Innovative strategies for personalized medicine: molecular approaches targeting the impact of inherited metabolic diseases caused by PPO mutations</a><br>
Continuities
P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)
Others
Publication year
2021
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
PLoS One
ISSN
1932-6203
e-ISSN
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Volume of the periodical
16
Issue of the periodical within the volume
11
Country of publishing house
US - UNITED STATES
Number of pages
15
Pages from-to
e0259837
UT code for WoS article
000755319200060
EID of the result in the Scopus database
2-s2.0-85119623124