Production of recombinant human deoxyribonuclease I in Luffa cylindrica L. and Nicotiana tabacum L.: evidence for protein secretion to the leaf intercellular space

Izabela Chincinska , Małgorzata Kapusta , Elżbieta Zielińska , Magdalena Miklaszewska , Kamila Błażejewska , Zbigniew Tukaj

Abstract

The sequence encoding hDNase I with its native N-terminal signal peptide was codon-optimized for heterological expression in cucurbit plants. The obtained synthetic gene rhDN was successfully transiently expressed in L. cylindrica (luffa) leaves using agroinfiltration. The results of in silico analysis suggest that the rhDN gene can be expressed in different cucurbits and in non-cucurbit species, such as a model plant Nicotiana tabacum. For further analysis of the recombinant hDNase I (rhDNase I) produced in planta we generated stably transformed tobacco lines (NtDN) overexpressing the rhDN gene. The presence of the active rhDNase I in the total protein extracts and in the intercellular fluids isolated from the leaves of the transgenic NtDN lines was demonstrated. However, the plants producing the rhDNase I did not show any visible changes in the phenotype. Using indirect immunofluorescence, we localized the rhDNase I in the intercellular space of NtDN leaves, which suggests that the native hDNase I signal peptide can be correctly recognized in the plant cells, what results in the protein targeting to the endoplasmic reticulum and then to the secretory pathway. Our results indicate the possibility to use the human DNase I as a molecular tool for improving and optimization of the plant expression systems based on cucurbits. Additionally, the established tobacco lines overexpressing the rhDN gene can be used as a platform to produce the active human DNase I.
Publication typeIn press (online first, early view)
Author Izabela Chincinska (FB / DPPB)
Izabela Chincinska,,
- Department of Plant Physiology and Biotechnology
, Małgorzata Kapusta (FB / DPCE)
Małgorzata Kapusta,,
- Department of Plant Cytology and Embryology
, Elżbieta Zielińska (FB / DPPB)
Elżbieta Zielińska,,
- Department of Plant Physiology and Biotechnology
, Magdalena Miklaszewska (FB / DPPB)
Magdalena Miklaszewska,,
- Department of Plant Physiology and Biotechnology
, Kamila Błażejewska (FB / DPPB)
Kamila Błażejewska,,
- Department of Plant Physiology and Biotechnology
, Zbigniew Tukaj (FB / DPPB)
Zbigniew Tukaj,,
- Department of Plant Physiology and Biotechnology
Journal seriesPlant Cell Tissue and Organ Culture, ISSN 0167-6857, (A 30 pkt)
Issue year2018
Noonline first
Pages1-13
Publication size in sheets0.6
Keywords in EnglishRecombinant human deoxyribonuclease I, tobacco overexpression system, Luffa, guttation fluid, recombinant proteins in an apoplast, agroinfiltration
DOIDOI:10.1007/s11240-018-1491-9
URL https://doi.org/10.1007/s11240-018-1491-9
Languageen angielski
Score (nominal)35
ScoreMinisterial score = 30.0, 16-11-2018, ArticleFromJournal
Ministerial score (2013-2016) = 35.0, 16-11-2018, ArticleFromJournal
Publication indicators WoS Impact Factor: 2016 = 2.002 (2) - 2016=2.001 (5)
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