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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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sp-Nef constructs are unstable in tobacco protoplasts. A: Protoplasts transfected with the indicated plasmids were pulse labelled for 2 h. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Note the size difference between cytosolic p25 and p27 (as indicated by arrowheads) and their signal-peptide fusion counterparts. Numbers at left indicate molecular weight markers in kDa. B: Protoplasts transfected with the indicated plasmids were pulse labelled for 1 h and chased for 5 h. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Arrowhead: aglycosylated Nef p25 (sp-p25Δg).
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Figure 4: sp-Nef constructs are unstable in tobacco protoplasts. A: Protoplasts transfected with the indicated plasmids were pulse labelled for 2 h. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Note the size difference between cytosolic p25 and p27 (as indicated by arrowheads) and their signal-peptide fusion counterparts. Numbers at left indicate molecular weight markers in kDa. B: Protoplasts transfected with the indicated plasmids were pulse labelled for 1 h and chased for 5 h. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Arrowhead: aglycosylated Nef p25 (sp-p25Δg).

Mentions: Having established that Nef is stable when expressed in the cytosol, we tested whether it was possible to express Nef in the secretory pathway. Therefore we fused the cDNAs for both p27 and p25 to the signal peptide (sp) of the PR1 protein [23] (constructs sp-p27 and sp-p25, Fig. 1). We have previously used this peptide successfully to drive the expression of immunoglobulin chains in tobacco cells [21]. We expressed the signal peptide fusions in tobacco protoplasts and subjected cells to 2 hours continuous labelling followed by homogenisation, immunoprecipitation with anti-FLAG antiserum, SDS-PAGE and fluorography. Figure 4A shows that both sp-p25 and sp-p27 are expressed and that their mobility is significantly reduced as compared to the cytosolic forms (Fig. 4A, note the size shift between lanes 1–2 and 3–4). Analysis of the Nef polypeptide sequence reveals the presence of two sequons that would be recognised as glycosylation sites upon translocation in the lumen of the endoplasmic reticulum. The presence of these glycans could explain the different electrophoretic mobility of the sp-Nef fusions. In order to prevent glycosylation – which of course does not occur during HIV infection – from interfering with the folding of the normally cytosolic, unglycosylated Nef protein, we inactivated the glycosylation sites by mutagenesis (constructs sp-p27Δgly and sp-p25 Δgly Fig. 1). We expressed these constructs in tobacco protoplasts and subjected cells to a 1-hour pulse labelling, followed by a 5-hour chase (Fig. 4B). Remarkably, both sp-p25 and sp-p27 resulted highly unstable, with their levels decreasing sharply during the 5-hour chase period (Fig. 4B, lanes 3–4 and 7–8). sp-p25Δgly showed increased mobility, as expected by removal of the glycosylation sites (Fig 4B, compare lanes 3–4 with lanes 5–6), but also disappeared during the chase. Expression of p27Δgly was barely detectable from the outset (Fig. 4B, lanes 9–10). None of the sp-Nef polypeptides appeared with time in the protoplast incubation medium (data not shown), indicating that sp-Nef fusions are capable of entering the secretory pathway (as indicated by glycosylation) but are not at all secreted. This phenotype – lack of secretion and fast intracellular degradation – indicates that the Nef polypeptides may be subject to strict quality control and subsequently disposed of by the secretory pathway [24-27]. Certainly the amount of 'secretory' Nef recovered after the chase did not compare favourably with the levels of cytosolically expressed protein. Targeting of Nef to the plant secretory pathway therefore proved to be a less successful strategy than cytosolic expression. For this reason we employed the cytosolic construct to generate stable transgenic tobacco plants.


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)

sp-Nef constructs are unstable in tobacco protoplasts. A: Protoplasts transfected with the indicated plasmids were pulse labelled for 2 h. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Note the size difference between cytosolic p25 and p27 (as indicated by arrowheads) and their signal-peptide fusion counterparts. Numbers at left indicate molecular weight markers in kDa. B: Protoplasts transfected with the indicated plasmids were pulse labelled for 1 h and chased for 5 h. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Arrowhead: aglycosylated Nef p25 (sp-p25Δg).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: sp-Nef constructs are unstable in tobacco protoplasts. A: Protoplasts transfected with the indicated plasmids were pulse labelled for 2 h. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Note the size difference between cytosolic p25 and p27 (as indicated by arrowheads) and their signal-peptide fusion counterparts. Numbers at left indicate molecular weight markers in kDa. B: Protoplasts transfected with the indicated plasmids were pulse labelled for 1 h and chased for 5 h. Cell homogenates were subjected to immunoprecipitation with anti-FLAG antiserum, followed by SDS-PAGE and fluorography. Arrowhead: aglycosylated Nef p25 (sp-p25Δg).
Mentions: Having established that Nef is stable when expressed in the cytosol, we tested whether it was possible to express Nef in the secretory pathway. Therefore we fused the cDNAs for both p27 and p25 to the signal peptide (sp) of the PR1 protein [23] (constructs sp-p27 and sp-p25, Fig. 1). We have previously used this peptide successfully to drive the expression of immunoglobulin chains in tobacco cells [21]. We expressed the signal peptide fusions in tobacco protoplasts and subjected cells to 2 hours continuous labelling followed by homogenisation, immunoprecipitation with anti-FLAG antiserum, SDS-PAGE and fluorography. Figure 4A shows that both sp-p25 and sp-p27 are expressed and that their mobility is significantly reduced as compared to the cytosolic forms (Fig. 4A, note the size shift between lanes 1–2 and 3–4). Analysis of the Nef polypeptide sequence reveals the presence of two sequons that would be recognised as glycosylation sites upon translocation in the lumen of the endoplasmic reticulum. The presence of these glycans could explain the different electrophoretic mobility of the sp-Nef fusions. In order to prevent glycosylation – which of course does not occur during HIV infection – from interfering with the folding of the normally cytosolic, unglycosylated Nef protein, we inactivated the glycosylation sites by mutagenesis (constructs sp-p27Δgly and sp-p25 Δgly Fig. 1). We expressed these constructs in tobacco protoplasts and subjected cells to a 1-hour pulse labelling, followed by a 5-hour chase (Fig. 4B). Remarkably, both sp-p25 and sp-p27 resulted highly unstable, with their levels decreasing sharply during the 5-hour chase period (Fig. 4B, lanes 3–4 and 7–8). sp-p25Δgly showed increased mobility, as expected by removal of the glycosylation sites (Fig 4B, compare lanes 3–4 with lanes 5–6), but also disappeared during the chase. Expression of p27Δgly was barely detectable from the outset (Fig. 4B, lanes 9–10). None of the sp-Nef polypeptides appeared with time in the protoplast incubation medium (data not shown), indicating that sp-Nef fusions are capable of entering the secretory pathway (as indicated by glycosylation) but are not at all secreted. This phenotype – lack of secretion and fast intracellular degradation – indicates that the Nef polypeptides may be subject to strict quality control and subsequently disposed of by the secretory pathway [24-27]. Certainly the amount of 'secretory' Nef recovered after the chase did not compare favourably with the levels of cytosolically expressed protein. Targeting of Nef to the plant secretory pathway therefore proved to be a less successful strategy than cytosolic expression. For this reason we employed the cytosolic construct to generate stable transgenic tobacco plants.

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