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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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| Published in: | PloS one 2021-11, Vol.16 (11), p.e0259837-e0259837 |
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| creator | Novakova, Zora Khuntsaria, Daria Gresova, Marketa Mikesova, Jana Havlinova, Barbora Shukla, Shivam Kolarova, Lucie Vesela, Katerina Martasek, Pavel Barinka, Cyril |
| 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. |
| doi_str_mv | 10.1371/journal.pone.0259837 |
| format | article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0259837</identifier><identifier>PMID: 34793488</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Agricultural production ; Analysis ; Animals ; Bacteria ; Biology ; Biology and Life Sciences ; Biosynthesis ; Biotechnology ; Cell Line ; Chromatography ; Cloning ; Comparative studies ; E coli ; Enzymes ; Escherichia coli - genetics ; Ethylenediaminetetraacetic acid ; Flavin-adenine dinucleotide ; Flavoproteins - biosynthesis ; Flavoproteins - genetics ; Flavoproteins - isolation & purification ; Gene expression ; Glycerol ; HEK293 Cells ; Heme ; Humans ; Impact analysis ; Insects ; Intracellular ; Laboratories ; Mammalian cells ; Mammals ; Metabolic disorders ; Mitochondrial Proteins - biosynthesis ; Mitochondrial Proteins - genetics ; Mitochondrial Proteins - isolation & purification ; Mutation ; Oxidase ; Oxygen ; Physical Sciences ; Plasmids ; Porphyria ; Post-translation ; Proteins ; Protoporphyrinogen oxidase ; Protoporphyrinogen Oxidase - biosynthesis ; Protoporphyrinogen Oxidase - genetics ; Protoporphyrinogen Oxidase - isolation & purification ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - isolation & purification ; Research and Analysis Methods ; Sf9 Cells</subject><ispartof>PloS one, 2021-11, Vol.16 (11), p.e0259837-e0259837</ispartof><rights>COPYRIGHT 2021 Public Library of Science</rights><rights>2021 Novakova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2021 Novakova et al 2021 Novakova et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-5f9e616aa347444905f545328b2e07e1ef7f89ab54629e54ae62800e00e69f3e3</citedby><cites>FETCH-LOGICAL-c692t-5f9e616aa347444905f545328b2e07e1ef7f89ab54629e54ae62800e00e69f3e3</cites><orcidid>0000-0001-9804-6346</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/2599054541/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/2599054541?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25753,27924,27925,37012,37013,44590,53791,53793,75126</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/34793488$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Millet, Oscar</contributor><creatorcontrib>Novakova, Zora</creatorcontrib><creatorcontrib>Khuntsaria, Daria</creatorcontrib><creatorcontrib>Gresova, Marketa</creatorcontrib><creatorcontrib>Mikesova, Jana</creatorcontrib><creatorcontrib>Havlinova, Barbora</creatorcontrib><creatorcontrib>Shukla, Shivam</creatorcontrib><creatorcontrib>Kolarova, Lucie</creatorcontrib><creatorcontrib>Vesela, Katerina</creatorcontrib><creatorcontrib>Martasek, Pavel</creatorcontrib><creatorcontrib>Barinka, Cyril</creatorcontrib><title>Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. 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biosynthesis</subject><subject>Mitochondrial Proteins - genetics</subject><subject>Mitochondrial Proteins - isolation & purification</subject><subject>Mutation</subject><subject>Oxidase</subject><subject>Oxygen</subject><subject>Physical Sciences</subject><subject>Plasmids</subject><subject>Porphyria</subject><subject>Post-translation</subject><subject>Proteins</subject><subject>Protoporphyrinogen oxidase</subject><subject>Protoporphyrinogen Oxidase - biosynthesis</subject><subject>Protoporphyrinogen Oxidase - genetics</subject><subject>Protoporphyrinogen Oxidase - isolation & purification</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Research and Analysis Methods</subject><subject>Sf9 Cells</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>PIMPY</sourceid><sourceid>DOA</sourceid><recordid>eNqNk12L1DAUhoso7rr6D0QLgujFjEnz0cYLYVjUHVhY8AvvQtqedLK0TU3SZebfm9npLlPZC2mh4fR53yRvcpLkJUZLTHL84dqOrlftcrA9LFHGREHyR8kpFiRb8AyRx0fjk-SZ99cIMVJw_jQ5ITQXhBbFaTJcQABnW9vY0aewHRx4b2yfqr5Oh9EZbSoV9gWrUweV7UrTqz6km7FTfTo4G-xg3bDZOdPbBiK3NbXykK5_f0xXaRQMykWHG0h9GOvd8-SJVq2HF9P3LPn55fOP84vF5dXX9fnqclFxkYUF0wI45krFpVJKBWKaUUayoswA5YBB57oQqmSUZwIYVcCzAiGILxeaADlLXh98h9Z6OYXlZcwpelFGcSTWB6K26loOznTK7aRVRt4WrGukcsFULUjMSkVpKUDhmgqNhcorritdZLiKOZbR69M021h2UFfQB6famen8T282srE3suAIM5RFg3eTgbN_RvBBdsZX0Laqh3gyt-vGBaWcRfTNP-jDu5uoRsUNmF7bOG-1N5UrXhBEWcFJpJYPUPGpoTNVvFnaxPpM8H4miEyAbWjU6L1cf__2_-zVrzn79ojdgGrDxtt23F89PwfpAayc9d6Bvg8ZI7lvjLs05L4x5NQYUfbq-IDuRXedQP4CfoAKTQ</recordid><startdate>20211118</startdate><enddate>20211118</enddate><creator>Novakova, Zora</creator><creator>Khuntsaria, Daria</creator><creator>Gresova, Marketa</creator><creator>Mikesova, Jana</creator><creator>Havlinova, Barbora</creator><creator>Shukla, Shivam</creator><creator>Kolarova, Lucie</creator><creator>Vesela, Katerina</creator><creator>Martasek, Pavel</creator><creator>Barinka, Cyril</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-9804-6346</orcidid></search><sort><creationdate>20211118</creationdate><title>Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study</title><author>Novakova, Zora ; Khuntsaria, Daria ; Gresova, Marketa ; Mikesova, Jana ; Havlinova, Barbora ; Shukla, Shivam ; Kolarova, Lucie ; Vesela, Katerina ; Martasek, Pavel ; Barinka, Cyril</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-5f9e616aa347444905f545328b2e07e1ef7f89ab54629e54ae62800e00e69f3e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Agricultural production</topic><topic>Analysis</topic><topic>Animals</topic><topic>Bacteria</topic><topic>Biology</topic><topic>Biology and Life Sciences</topic><topic>Biosynthesis</topic><topic>Biotechnology</topic><topic>Cell Line</topic><topic>Chromatography</topic><topic>Cloning</topic><topic>Comparative studies</topic><topic>E coli</topic><topic>Enzymes</topic><topic>Escherichia coli - 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>34793488</pmid><doi>10.1371/journal.pone.0259837</doi><tpages>e0259837</tpages><orcidid>https://orcid.org/0000-0001-9804-6346</orcidid><oa>free_for_read</oa></addata></record> |
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| recordid | cdi_plos_journals_2599054541 |
| source | Publicly Available Content Database (Proquest) (PQ_SDU_P3); PubMed Central Free |
| subjects | Agricultural production Analysis Animals Bacteria Biology Biology and Life Sciences Biosynthesis Biotechnology Cell Line Chromatography Cloning Comparative studies E coli Enzymes Escherichia coli - genetics Ethylenediaminetetraacetic acid Flavin-adenine dinucleotide Flavoproteins - biosynthesis Flavoproteins - genetics Flavoproteins - isolation & purification Gene expression Glycerol HEK293 Cells Heme Humans Impact analysis Insects Intracellular Laboratories Mammalian cells Mammals Metabolic disorders Mitochondrial Proteins - biosynthesis Mitochondrial Proteins - genetics Mitochondrial Proteins - isolation & purification Mutation Oxidase Oxygen Physical Sciences Plasmids Porphyria Post-translation Proteins Protoporphyrinogen oxidase Protoporphyrinogen Oxidase - biosynthesis Protoporphyrinogen Oxidase - genetics Protoporphyrinogen Oxidase - isolation & purification Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Research and Analysis Methods Sf9 Cells |
| title | Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study |
| url | http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-04T13%3A46%3A05IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Heterologous%20expression%20and%20purification%20of%20recombinant%20human%20protoporphyrinogen%20oxidase%20IX:%20A%20comparative%20study&rft.jtitle=PloS%20one&rft.au=Novakova,%20Zora&rft.date=2021-11-18&rft.volume=16&rft.issue=11&rft.spage=e0259837&rft.epage=e0259837&rft.pages=e0259837-e0259837&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0259837&rft_dat=%3Cgale_plos_%3EA683045863%3C/gale_plos_%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-c692t-5f9e616aa347444905f545328b2e07e1ef7f89ab54629e54ae62800e00e69f3e3%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_pqid=2599054541&rft_id=info:pmid/34793488&rft_galeid=A683045863&rfr_iscdi=true |