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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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Nef is expressed and is stable in the cytosol of tobacco protoplasts. Tobacco mesophyll protoplasts were transfected with plasmids encoding the p27, p25 and p27 mut Nef variants, or with empty vector (Co). Transfected cells were labelled for 1 h with 35S methionine and cysteine and chased for 5 h with unlabelled amino acids. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Numbers at left indicate molecular weight markers in kDa.
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Figure 2: Nef is expressed and is stable in the cytosol of tobacco protoplasts. Tobacco mesophyll protoplasts were transfected with plasmids encoding the p27, p25 and p27 mut Nef variants, or with empty vector (Co). Transfected cells were labelled for 1 h with 35S methionine and cysteine and chased for 5 h with unlabelled amino acids. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Numbers at left indicate molecular weight markers in kDa.

Mentions: We set out to evaluate the ability of plant cells to cope with the synthesis of Nef. We generated a number of constructs for Nef expression in the plant cytosol or secretory pathway (Fig. 1). To facilitate immunodetection of the viral polypeptides, all constructs bore the FLAG and 6× histidine epitope tags at their C-termini, with the exception of wild-type p27 Nef that only carried the FLAG tag. We tested these constructs by transient expression in tobacco mesophyll protoplasts followed by pulse-chase analysis. We initially expressed the cytosolic forms of Nef: full length, both with (p27) or without (p27 mut) its N-terminal myristoylation signal, and truncated form (p25). Transfected protoplasts were metabolically labelled with 35S-methionine and 35S-cysteine and chased for 5 hours. Protoplast homogenates were then subjected to immunoprecipitation with anti FLAG antiserum. SDS-PAGE and fluorography revealed that all three constructs are successfully expressed, yielding polypeptides of the expected sizes (Fig. 2). Note that p27 is faster migrating than p27 mut as it does not contain the additional histidine tag (Fig. 2, compare lanes 4–5 with lanes 2–3). All proteins appeared to be stable over the course of the 5-hour chase.


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)

Nef is expressed and is stable in the cytosol of tobacco protoplasts. Tobacco mesophyll protoplasts were transfected with plasmids encoding the p27, p25 and p27 mut Nef variants, or with empty vector (Co). Transfected cells were labelled for 1 h with 35S methionine and cysteine and chased for 5 h with unlabelled amino acids. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. 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 2: Nef is expressed and is stable in the cytosol of tobacco protoplasts. Tobacco mesophyll protoplasts were transfected with plasmids encoding the p27, p25 and p27 mut Nef variants, or with empty vector (Co). Transfected cells were labelled for 1 h with 35S methionine and cysteine and chased for 5 h with unlabelled amino acids. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Numbers at left indicate molecular weight markers in kDa.
Mentions: We set out to evaluate the ability of plant cells to cope with the synthesis of Nef. We generated a number of constructs for Nef expression in the plant cytosol or secretory pathway (Fig. 1). To facilitate immunodetection of the viral polypeptides, all constructs bore the FLAG and 6× histidine epitope tags at their C-termini, with the exception of wild-type p27 Nef that only carried the FLAG tag. We tested these constructs by transient expression in tobacco mesophyll protoplasts followed by pulse-chase analysis. We initially expressed the cytosolic forms of Nef: full length, both with (p27) or without (p27 mut) its N-terminal myristoylation signal, and truncated form (p25). Transfected protoplasts were metabolically labelled with 35S-methionine and 35S-cysteine and chased for 5 hours. Protoplast homogenates were then subjected to immunoprecipitation with anti FLAG antiserum. SDS-PAGE and fluorography revealed that all three constructs are successfully expressed, yielding polypeptides of the expected sizes (Fig. 2). Note that p27 is faster migrating than p27 mut as it does not contain the additional histidine tag (Fig. 2, compare lanes 4–5 with lanes 2–3). All proteins appeared to be stable over the course of the 5-hour chase.

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