Production of recombinant human deoxyribonuclease I in Luffa cylindrica L. and Nicotiana tabacum L.: evidence for protein secretion to the leaf intercellular space
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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.
KeywordsRecombinant human deoxyribonuclease I Tobacco overexpression system Luffa Guttation fluid Recombinant proteins in an apoplast Agroinfiltration
We gratefully acknowledge Dr. hab. Christina Kühn and Dr hab. Stefan Tukaj for the sincere support.
This research was supported by the Foundation for Polish Science (FNP, POMOST/2011-4/4) co-financed from the European Union under the European Regional Development Fund.
Compliance with ethical standards
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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