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Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells.

TitoloExpression, intracellular targeting and purification of HIV Nef variants in tobacco cells.
Tipo di pubblicazioneArticolo su Rivista peer-reviewed
Anno di Pubblicazione2007
AutoriMarusic, Carla, Nuttall James, Buriani Giampaolo, Lico Chiara, Lombardi Raffaele, Baschieri Selene, Benvenuto Eugenio, and Frigerio Lorenzo
RivistaBMC Biotechnol
Volume7
Paginazione12
Data di pubblicazione2007 Feb 26
Parole chiaveCells, Cultured, Drug Delivery Systems, Gene Products, nef, nef Gene Products, Human Immunodeficiency Virus, Plants, Genetically Modified, protein engineering, Recombinant Proteins, Subcellular Fractions, Tobacco
Abstract

BACKGROUND: Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco.

RESULTS: We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure.

CONCLUSION: We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

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cited By 41

URLhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-33847788546&doi=10.1186%2f1472-6750-7-12&partnerID=40&md5=8d996f09742baff76b96939caa3e76dd
DOI10.1186/1472-6750-7-12
Alternate JournalBMC Biotechnol.
Citation Key6045
PubMed ID17324250
PubMed Central IDPMC1808453