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

Marusic C, Nuttall J, Buriani G, Lico C, Lombardi R, Baschieri S, Benvenuto E, Frigerio L - BMC Biotechnol. (2007)

Bottom Line: 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).We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants.The proteins can easily be purified from transgenic tissue.

View Article: PubMed Central - HTML - PubMed

Affiliation: ENEA-BIOTEC Sezione Genetica e Genomica Vegetale, C.R. Casaccia, 00060 Rome, Italy. carla.marusic@casaccia.enea.it <carla.marusic@casaccia.enea.it>

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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ELISA analysis of transgenic tobacco plants expressing cytosolic Nef. A:Representative ELISA assay of p27 mut Nef tobacco lines. C+: 100 ng of E.coli recombinant Nef; C-: control plant, untrasformed tobacco. The numbers on the x axis indicate individual transgenic tobacco lines. B:Representative ELISA assay of p25 Nef tobacco lines. C+: 100 ng of E.coli recombinant Nef; C-: control plant, untrasformed tobacco. The numbers on the x axis indicate individual transgenic tobacco lines. C: ELISA quantification of plant-expressed Nef variants. The Nef expression levels are expressed as percentage of total soluble protein (TSP) from transgenic plants. The values shown represent averages of two experiments. The numbers on the x axis indicate individual transgenic tobacco lines.
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Figure 5: ELISA analysis of transgenic tobacco plants expressing cytosolic Nef. A:Representative ELISA assay of p27 mut Nef tobacco lines. C+: 100 ng of E.coli recombinant Nef; C-: control plant, untrasformed tobacco. The numbers on the x axis indicate individual transgenic tobacco lines. B:Representative ELISA assay of p25 Nef tobacco lines. C+: 100 ng of E.coli recombinant Nef; C-: control plant, untrasformed tobacco. The numbers on the x axis indicate individual transgenic tobacco lines. C: ELISA quantification of plant-expressed Nef variants. The Nef expression levels are expressed as percentage of total soluble protein (TSP) from transgenic plants. The values shown represent averages of two experiments. The numbers on the x axis indicate individual transgenic tobacco lines.

Mentions: We analysed the levels of Nef protein expression by direct ELISA, using as primary antibody the antiserum to HIV-1 Nef (Fig. 5A–B). In these assays E.coli recombinant Nef was used as positive control. Fifteen p27 mut and 6 p25 lines were found to express Nef variants in detectable amounts. Among these, 3 lines expressing p27 mut (Fig. 5A, lines 64, 112, 116) and two lines expressing p25 (Fig. 5B, lines 79, 120) were further analysed to estimate Nef expression levels. We estimated the overall amounts of plant recombinant Nef – expressed as percentage of total soluble plant protein (TSP) – by using a standard curve generated with different concentrations of E.coli-produced recombinant Nef. As shown in Fig. 5C, the quantitative ELISA revealed variable expression levels of p25 and p27 mut Nef variants in TSP from individual transgenic lines ranging between 0.18% of TSP (Fig. 5C, p27 mut-116) and 0.7% (Fig. 5C, p25–79).


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

Marusic C, Nuttall J, Buriani G, Lico C, Lombardi R, Baschieri S, Benvenuto E, Frigerio L - BMC Biotechnol. (2007)

ELISA analysis of transgenic tobacco plants expressing cytosolic Nef. A:Representative ELISA assay of p27 mut Nef tobacco lines. C+: 100 ng of E.coli recombinant Nef; C-: control plant, untrasformed tobacco. The numbers on the x axis indicate individual transgenic tobacco lines. B:Representative ELISA assay of p25 Nef tobacco lines. C+: 100 ng of E.coli recombinant Nef; C-: control plant, untrasformed tobacco. The numbers on the x axis indicate individual transgenic tobacco lines. C: ELISA quantification of plant-expressed Nef variants. The Nef expression levels are expressed as percentage of total soluble protein (TSP) from transgenic plants. The values shown represent averages of two experiments. The numbers on the x axis indicate individual transgenic tobacco lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808453&req=5

Figure 5: ELISA analysis of transgenic tobacco plants expressing cytosolic Nef. A:Representative ELISA assay of p27 mut Nef tobacco lines. C+: 100 ng of E.coli recombinant Nef; C-: control plant, untrasformed tobacco. The numbers on the x axis indicate individual transgenic tobacco lines. B:Representative ELISA assay of p25 Nef tobacco lines. C+: 100 ng of E.coli recombinant Nef; C-: control plant, untrasformed tobacco. The numbers on the x axis indicate individual transgenic tobacco lines. C: ELISA quantification of plant-expressed Nef variants. The Nef expression levels are expressed as percentage of total soluble protein (TSP) from transgenic plants. The values shown represent averages of two experiments. The numbers on the x axis indicate individual transgenic tobacco lines.
Mentions: We analysed the levels of Nef protein expression by direct ELISA, using as primary antibody the antiserum to HIV-1 Nef (Fig. 5A–B). In these assays E.coli recombinant Nef was used as positive control. Fifteen p27 mut and 6 p25 lines were found to express Nef variants in detectable amounts. Among these, 3 lines expressing p27 mut (Fig. 5A, lines 64, 112, 116) and two lines expressing p25 (Fig. 5B, lines 79, 120) were further analysed to estimate Nef expression levels. We estimated the overall amounts of plant recombinant Nef – expressed as percentage of total soluble plant protein (TSP) – by using a standard curve generated with different concentrations of E.coli-produced recombinant Nef. As shown in Fig. 5C, the quantitative ELISA revealed variable expression levels of p25 and p27 mut Nef variants in TSP from individual transgenic lines ranging between 0.18% of TSP (Fig. 5C, p27 mut-116) and 0.7% (Fig. 5C, p25–79).

Bottom Line: 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).We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants.The proteins can easily be purified from transgenic tissue.

View Article: PubMed Central - HTML - PubMed

Affiliation: ENEA-BIOTEC Sezione Genetica e Genomica Vegetale, C.R. Casaccia, 00060 Rome, Italy. carla.marusic@casaccia.enea.it <carla.marusic@casaccia.enea.it>

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.

Show MeSH
Related in: MedlinePlus