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2023-02-21Zeitschriftenartikel DOI: 10.18452/26261
A CRR2-Dependent sRNA Sequence Supports Papillomavirus Vaccine Expression in Tobacco Chloroplasts
dc.contributor.authorLegen, Julia
dc.contributor.authorDühnen, Sara
dc.contributor.authorGauert, Anton
dc.contributor.authorGötz, Michael
dc.contributor.authorSchmitz-Linneweber, Christian
dc.contributor.editorDemurtas, Olivia Costantina
dc.contributor.editorFrusciante, Sarah
dc.date.accessioned2023-03-20T14:07:39Z
dc.date.available2023-03-20T14:07:39Z
dc.date.issued2023-02-21none
dc.date.updated2023-03-08T04:15:09Z
dc.identifier.urihttp://edoc.hu-berlin.de/18452/26935
dc.description.abstractIntroduction: Human papillomavirus (HPV) infection is the leading cause of cervical cancer, and vaccination with HPV L1 capsid proteins has been successful in controlling it. However, vaccination coverage is not universal, particularly in developing countries, where 80% of all cervical cancer cases occur. Cost-effective vaccination could be achieved by expressing the L1 protein in plants. Various efforts have been made to produce the L1 protein in plants, including attempts to express it in chloroplasts for high-yield performance. However, manipulating chloroplast gene expression requires complex and difficult-to-control expression elements. In recent years, a family of nuclear-encoded, chloroplast-targeted RNA-binding proteins, the pentatricopeptide repeat (PPR) proteins, were described as key regulators of chloroplast gene expression. For example, PPR proteins are used by plants to stabilize and translate chloroplast mRNAs. Objectives: To demonstrate that a PPR target site can be used to drive HPV L1 expression in chloroplasts. Methods: To test our hypothesis, we used biolistic chloroplast transformation to establish tobacco lines that express two variants of the HPV L1 protein under the control of the target site of the PPR protein CHLORORESPIRATORY REDUCTION2 (CRR2). The transgenes were inserted into a dicistronic operon driven by the plastid rRNA promoter. To determine the effectiveness of the PPR target site for the expression of the HPV L1 protein in the chloroplasts, we analyzed the accumulation of the transgenic mRNA and its processing, as well as the accumulation of the L1 protein in the transgenic lines. Results: We established homoplastomic lines carrying either the HPV18 L1 protein or an HPV16B Enterotoxin::L1 fusion protein. The latter line showed severe growth retardation and pigment loss, suggesting that the fusion protein is toxic to the chloroplasts. Despite the presence of dicistronic mRNAs, we observed very little accumulation of monocistronic transgenic mRNA and no significant increase in CRR2-associated small RNAs. Although both lines expressed the L1 protein, quantification using an external standard suggested that the amounts were low. Conclusions: Our results suggest that PPR binding sites can be used to drive vaccine expression in plant chloroplasts; however, the factors that modulate the effectiveness of target gene expression remain unclear. The identification of dozens of PPR binding sites through small RNA sequencing expands the set of expression elements available for high-value protein production in chloroplasts.eng
dc.description.sponsorshipGerman Research Foundation, DFG
dc.language.isoengnone
dc.publisherHumboldt-Universität zu Berlin
dc.rights(CC BY 4.0) Attribution 4.0 Internationalger
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectchloroplasteng
dc.subjecttransgene expressioneng
dc.subjectRNA processingeng
dc.subjectRNA bindingeng
dc.subjectPPReng
dc.subjectpentatricopeptide repeateng
dc.subjectRNA stabilityeng
dc.subjectorganelleeng
dc.subjectNicotiana tabacumeng
dc.subject.ddc610 Medizin und Gesundheitnone
dc.titleA CRR2-Dependent sRNA Sequence Supports Papillomavirus Vaccine Expression in Tobacco Chloroplastsnone
dc.typearticle
dc.identifier.urnurn:nbn:de:kobv:11-110-18452/26935-5
dc.identifier.doihttp://dx.doi.org/10.18452/26261
dc.type.versionpublishedVersionnone
local.edoc.pages13none
local.edoc.type-nameZeitschriftenartikel
local.edoc.container-typeperiodical
local.edoc.container-type-nameZeitschrift
dc.description.versionPeer Reviewednone
dc.identifier.eissn2218-1989
dcterms.bibliographicCitation.doi10.3390/metabo13030315none
dcterms.bibliographicCitation.journaltitleMetabolitesnone
dcterms.bibliographicCitation.volume13none
dcterms.bibliographicCitation.issue3none
dcterms.bibliographicCitation.articlenumber315none
dcterms.bibliographicCitation.originalpublishernameMDPInone
dcterms.bibliographicCitation.originalpublisherplaceBaselnone
bua.departmentLebenswissenschaftliche Fakultätnone

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