title: Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: ENEA-BIOTEC Sezione Genetica e Genomica Vegetale, C.R. Casaccia, 00060 Rome, Italy. carla.marusic@casaccia.enea.it <carla.marusic@casaccia.enea.it>
authors.forename: Carla
authors.initials: C
authors.lastname: Marusic
authors.forename: James
authors.initials: J
authors.lastname: Nuttall
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Buriani
authors.forename: Chiara
authors.initials: C
authors.lastname: Lico
authors.forename: Raffaele
authors.initials: R
authors.lastname: Lombardi
authors.forename: Selene
authors.initials: S
authors.lastname: Baschieri
authors.forename: Eugenio
authors.initials: E
authors.lastname: Benvenuto
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Frigerio
mesh_terms: D002478:Cells, Cultured; D000090266:Chromobox Protein Homolog 5; D016503:Drug Delivery Systems / Q000379:methods; D015693:Gene Products, nef / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D030821:Plants, Genetically Modified / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013347:Subcellular Fractions / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus
chemical_list: C000715594:CBX5 protein, human; D015693:Gene Products, nef; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D000090266:Chromobox Protein Homolog 5