Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study.

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...

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Autores principales:	Zora Novakova, Daria Khuntsaria, Marketa Gresova, Jana Mikesova, Barbora Havlinova, Shivam Shukla, Lucie Kolarova, Katerina Vesela, Pavel Martasek, Cyril Barinka
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/496d160f1f0648b29aead47a6c558c56
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spelling	oai:doaj.org-article:496d160f1f0648b29aead47a6c558c562021-12-02T20:12:50ZHeterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study.1932-620310.1371/journal.pone.0259837https://doaj.org/article/496d160f1f0648b29aead47a6c558c562021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0259837https://doaj.org/toc/1932-6203Human 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.Zora NovakovaDaria KhuntsariaMarketa GresovaJana MikesovaBarbora HavlinovaShivam ShuklaLucie KolarovaKaterina VeselaPavel MartasekCyril BarinkaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 11, p e0259837 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Zora Novakova Daria Khuntsaria Marketa Gresova Jana Mikesova Barbora Havlinova Shivam Shukla Lucie Kolarova Katerina Vesela Pavel Martasek Cyril Barinka Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study.
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.
format	article
author	Zora Novakova Daria Khuntsaria Marketa Gresova Jana Mikesova Barbora Havlinova Shivam Shukla Lucie Kolarova Katerina Vesela Pavel Martasek Cyril Barinka
author_facet	Zora Novakova Daria Khuntsaria Marketa Gresova Jana Mikesova Barbora Havlinova Shivam Shukla Lucie Kolarova Katerina Vesela Pavel Martasek Cyril Barinka
author_sort	Zora Novakova
title	Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study.
title_short	Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study.
title_full	Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study.
title_fullStr	Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study.
title_full_unstemmed	Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study.
title_sort	heterologous expression and purification of recombinant human protoporphyrinogen oxidase ix: a comparative study.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/496d160f1f0648b29aead47a6c558c56
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Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study.

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