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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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Analysis and affinity purification of plant-produced cytosolic Nef. A: Samples of leaf protein homogenates from a p25 (line 120) and a p27 mut (line 116) transgenic line were resolved by 15% SDS-PAGE and subjected to immunoblot with anti-FLAG polyclonal antiserum. A dilution series of a recombinant, FLAG-tagged ScFv [28] was loaded as a quantitative reference. The asterisk indicates a non-specific, anti FLAG – immunoreactive peptide. Numbers at left indicate molecular weight markers in kDa. Co: control mock-transformed plant. B: Total leaf protein homogenate from transgenic plant line 116 expressing p27 mut was subjected to affinity chromatography with a cobalt resin. His-tagged Nef was eluted with imidazole. U: fraction not bound to the resin; W: wash fraction. Numbers at left indicate molecular weight markers in kDa.
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Figure 6: Analysis and affinity purification of plant-produced cytosolic Nef. A: Samples of leaf protein homogenates from a p25 (line 120) and a p27 mut (line 116) transgenic line were resolved by 15% SDS-PAGE and subjected to immunoblot with anti-FLAG polyclonal antiserum. A dilution series of a recombinant, FLAG-tagged ScFv [28] was loaded as a quantitative reference. The asterisk indicates a non-specific, anti FLAG – immunoreactive peptide. Numbers at left indicate molecular weight markers in kDa. Co: control mock-transformed plant. B: Total leaf protein homogenate from transgenic plant line 116 expressing p27 mut was subjected to affinity chromatography with a cobalt resin. His-tagged Nef was eluted with imidazole. U: fraction not bound to the resin; W: wash fraction. Numbers at left indicate molecular weight markers in kDa.

Mentions: In order to assess the integrity of the plant-expressed Nef polypeptides, we collected tissue samples from plants expressing p25 or p27 mut and subjected them to SDS-PAGE alongside increasing concentrations of a FLAG-tagged anti-lysozyme ScFv [28] as a quantitative control (Fig. 6A). Immunoblot with anti FLAG antiserum revealed an immunoreactive polypeptide of the size predicted for both p25 and p27 mut and showed clear accumulation of the proteins. In addition, a large amount of faster migrating immunoreactive peptides were also detected. As these are not present in samples from control plants containing empty vector, they are likely to be degradation products of the Nef polypeptide.


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)

Analysis and affinity purification of plant-produced cytosolic Nef. A: Samples of leaf protein homogenates from a p25 (line 120) and a p27 mut (line 116) transgenic line were resolved by 15% SDS-PAGE and subjected to immunoblot with anti-FLAG polyclonal antiserum. A dilution series of a recombinant, FLAG-tagged ScFv [28] was loaded as a quantitative reference. The asterisk indicates a non-specific, anti FLAG – immunoreactive peptide. Numbers at left indicate molecular weight markers in kDa. Co: control mock-transformed plant. B: Total leaf protein homogenate from transgenic plant line 116 expressing p27 mut was subjected to affinity chromatography with a cobalt resin. His-tagged Nef was eluted with imidazole. U: fraction not bound to the resin; W: wash fraction. Numbers at left indicate molecular weight markers in kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Analysis and affinity purification of plant-produced cytosolic Nef. A: Samples of leaf protein homogenates from a p25 (line 120) and a p27 mut (line 116) transgenic line were resolved by 15% SDS-PAGE and subjected to immunoblot with anti-FLAG polyclonal antiserum. A dilution series of a recombinant, FLAG-tagged ScFv [28] was loaded as a quantitative reference. The asterisk indicates a non-specific, anti FLAG – immunoreactive peptide. Numbers at left indicate molecular weight markers in kDa. Co: control mock-transformed plant. B: Total leaf protein homogenate from transgenic plant line 116 expressing p27 mut was subjected to affinity chromatography with a cobalt resin. His-tagged Nef was eluted with imidazole. U: fraction not bound to the resin; W: wash fraction. Numbers at left indicate molecular weight markers in kDa.
Mentions: In order to assess the integrity of the plant-expressed Nef polypeptides, we collected tissue samples from plants expressing p25 or p27 mut and subjected them to SDS-PAGE alongside increasing concentrations of a FLAG-tagged anti-lysozyme ScFv [28] as a quantitative control (Fig. 6A). Immunoblot with anti FLAG antiserum revealed an immunoreactive polypeptide of the size predicted for both p25 and p27 mut and showed clear accumulation of the proteins. In addition, a large amount of faster migrating immunoreactive peptides were also detected. As these are not present in samples from control plants containing empty vector, they are likely to be degradation products of the Nef polypeptide.

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