Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells
- Carla Marusic†1,
- James Nuttall†2,
- Giampaolo Buriani1,
- Chiara Lico1,
- Raffaele Lombardi1,
- Selene Baschieri1,
- Eugenio Benvenuto1Email author and
- Lorenzo Frigerio2Email author
© Marusic et al; licensee BioMed Central Ltd. 2007
Received: 01 November 2006
Accepted: 26 February 2007
Published: 26 February 2007
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.
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.
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.
Plants have emerged as a safe and economical alternative to mainstream protein expression systems based on the large-scale culture of microbes or animal cells or on transgenic animals to produce biopharmaceuticals. Diverse, complex macromolecules such as antibodies [1, 2] and vaccine components  have been successfully expressed in plant cells. The possibility to produce biopharmaceuticals using plants offers solutions to some of the problems associated to traditional heterologous expression systems. For example, the bacterial production of biologically active, complex multimeric proteins such as antibodies is limited by the absence of the enzymatic machinery involved in post-translational modification of newly-synthesised proteins [1, 2]. Among eukaryotic expression systems, yeast is not always appropriate as hyperglycosylation of the final product is often encountered, even if several laboratories are in the process of modulating glycosylation pathways to obtain humanized yeast-derived glycoproteins [4, 5]. Insect and mammalian cell cultures represent complex expression platforms requiring expensive procedures and may be easily contaminated with toxins, viruses or prions, raising concerns on the safety of the final product. The plant secretory pathway, on the other hand, has been shown to be particularly suitable for the production and accumulation of high amounts of heterologous proteins [6, 7].
Modern vaccines are becoming increasingly complex, with several constituted by a combination of multiple antigens. Most of the current strategies for vaccination against HIV/AIDS involve targeting a combination of HIV and host antigens . Plant-based expression of a number of these candidates has already been achieved, including HIV-1 gp120 envelope glycoprotein , p24 core protein  and the regulatory Tat protein .
Both regulatory and accessory HIV proteins are currently regarded as promising targets for vaccine development as they could provide further protective efficacy in combination with viral structural proteins. For this purpose, HIV-1 accessory Nef protein is considered a promising target for vaccine development .
Nef is incorporated into viral particles and expressed in the early stage of infection both in the cytoplasm and on the cell membrane of virus-infected cells. Nef interacts with multiple host factors in order to optimise the cellular environment for virus replication . Its critical role for viral pathogenicity is demonstrated by the fact that the infection with nef-defective HIV strains dramatically decreases the rate of disease progression in seropositive individuals . Moreover, Nef is an important component for CTL-based HIV-1 vaccines. For this reason immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading .
In vitro proteolysis experiments have shown that Nef consists of an N-terminal membrane anchor region and a well folded C-terminal core domain . The N-terminal membrane anchor domain structure has been solved in its myristoylated and non-myristoylated forms showing a flexible polypeptide chain with two helical structure elements .
When translated in vitro, the Nef gene yields two main polypeptides: a full-length N-terminal myristoylated form of 27 kDa (p27) and a truncated form of 25 kDa (p25) translated from a second start codon of the Nef gene and lacking the first 18 amino acids. Non-myristoylated p27 Nef mutant and p25 Nef were both found in the cytoplasm, while the wild-type, presumably myristoylated p27 Nef was mainly membrane associated . Both p27 and p25 have been expressed in different biological systems. While the levels of p27 non-myristoylated expression in E. coli are reasonably high , protein yield in yeast and insect cells is very poor . In particular, from the analysis of subcellular localization of the recombinant protein in yeast, it appears that the myristoylated form of Nef causes cell membranes perturbation . Moreover, it has been shown that Nef expressed in transfected mammalian cell lines can be cytotoxic and cytostatic .
To explore the possibility of Nef expression in plants, we attempted a number of different strategies. We designed a panel of constructs to direct the expression of Nef polypeptides to either the cytosol or the secretory pathway. We tested these constructs initially by transient expression in tobacco protoplasts, to rapidly ascertain the most promising strategy for Nef production in stable transgenic plants. Plant protoplast transfection is an established in vivo approach that allows rapid assessment of heterologous protein expression in plant cells . Moreover it allows for an accurate assessment of the intracellular fate of cytosolic or ER targeted recombinant proteins . Here, we report the expression of the p25 and p27 HIV Nef polypeptides and of their non-myristoylated p27 variants.
Nef is stable in the plant cytosol
Nef is unstable in the plant secretory pathway
Stable Nef expression in transgenic tobacco and affinity purification
The sequences encoding p25 and p27 mut HIV-1 Nef variants were cloned into the binary plant expression vector pBI121 and the constructs p25 and p27 mut (Fig.1) used to generate transgenic tobacco plants. The putative transgenic tobacco plants were analysed by polymerase chain reaction (PCR) to verify the nuclear integration of nef gene (data not shown). Seventy p25 and 118 p27 mut lines were analysed, among these 67 and 115 respectively were positive for nef gene (data not shown).
We took advantage of the 6× histidine tag appended to p27 mut to perform a small-scale purification of Nef by loading homogenates from transgenic leaf sections on a cobalt affinity purification column. Immunoblot with anti FLAG antiserum (Fig. 6B) shows that the eluate from this one-step procedure yields an anti FLAG-immunoreactive polypeptide of the size expected for p27 mut. No degradation products are detected, indicating that the purified protein is stable.
Discussion and conclusion
We have assessed a number of strategies for the expression of HIV Nef in planta. We initially used tobacco mesophyll protoplast transfection to test constructs encoding full-length Nef, with (p27) or without its myristoylation signal (p27 mut), or truncated Nef (p25). The use of protoplast transfection, coupled with metabolic labelling and pulse-chase experiments, allowed us to very rapidly determine what intracellular location was most suitable for expression of Nef. Our results indicate that cytosolic Nef polypeptides are correctly synthesised and relatively stable over a 5-hour time course. The same was not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. Although the proteins translocated correctly into the lumen of the endoplasmic reticulum, as demonstrated by efficient N-glycosylation, they were not stable. Moreover, there was no evidence for secretion of any of the proteins. Even the removal of the glycosylation sites did not significantly improve stability, or promote secretion. Although redirection of cytosolic proteins into the lumen of the secretory pathway has often achieved high yield of recombinant protein [29–32] it is possible that Nef, which is normally located in the cytosol or in association with the cytosolic face of the plasma membrane , does not fold correctly within the milieu of the endoplasmic reticulum. The fact that no polypeptides are secreted and that degradation seems to occur very rapidly indicate that Nef may be disposed of by ER quality control mechanisms [26, 33].
Our data indicate that cytosolic expression is a more promising strategy. We therefore generated transgenic plants expressing full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. This is in line with a number of other heterologous proteins expressed in transgenic tobacco . Moreover, it is possible that the ELISA values represent an underestimate. We have indeed found that plant protein homogenates partially interfere with the detection of recombinant Nef in ELISA assay: by mixing recombinant E.coli HIV-1 Nef with control plant protein extracts, we often observed a significant reduction in the ELISA reading as compared with the Nef protein in buffer alone (data not shown).
Interestingly, cytosolically expressed Nef is almost completely non-myristoylated, as indicated by poor membrane partitioning of Nef p27. This is somewhat surprising, as the N-terminal myristoylation machinery is conserved in plants [35, 36]. In any case, the lack of myristoylation is actually beneficial to the immunogenic properties of Nef for the application in a multi-component vaccine . Indeed, deletion or mutagenesis of the N-terminal myristoylation site has been shown to abrogate the capacity of Nef to down-regulate both MHC class I and CD4 cell-surface molecules , which normally prevents CTL-mediated lysis of HIV-1-primary infected cells . Therefore the lack of myristoylation is likely to elicit enhanced cellular immune responses.
Plant-expressed Nef could be purified easily from transgenic leaves using a cobalt affinity column. This indicates that the hexahistidine tag is correctly exposed in the plant-made polypeptides. This ease of purification, together with our data showing that cytosolic Nef expression levels in planta are satisfactory, constitutes a useful starting point for further optimisation and scale-up of expression. This will allow us to analyse the biological activity and in vitro/in vivo immunological properties of plant-produced Nef proteins.
The pSCNef51 (ARP#2015 NIBSC-CFAR MRC) plasmid was used as the source of Nef cDNA (HIV-1 BH10 strain). In all constructs (Figure 1) Nef encoding sequences were under the control of the constitutive CaMV 35S promoter and the nopaline synthase (NOS) terminator sequence. To enhance translational efficiency, the Tobacco Mosaic Virus (TMV) 5' leader sequence Ω  was fused in frame with the Nef sequences. Moreover to facilitate the purification and detection of the recombinant proteins, all Nef coding sequences harbour at their 3'end the sequences encoding the FLAG and polyhistidine-tag peptides, with the exception of wild-type p27 Nef that only carried the FLAG tag. All inserts were generated by overlap extension PCR . Briefly, a set of primers was used to generate by PCR, using Pfu polymerase (Stratagene, La Jolla, CA), two DNA fragments having overlapping ends. PCR-generated DNA fragments were first purified from agarose gel using GFX PCR DNA and Gel Band Purification Kit (Amersham Bioscience) and then employed in a subsequent overlap extension reaction to obtain the resulting fusion products. To amplify the fusion products cloned in the constructs p25 and p27 mut (Figure 1) the following forward primers were used BNFor (5' CGGGATCCATGAGACGAGCTGAGCCAGCAGCAGATG 3') and BNMFor (5' CGGGATCCATGGCCGGCAAGTGGTCAAAAAGTAGTGTG 3'). The HSRev (5' TCCCCCGGGCTAATGGTGATGGTGATGGTGCTTGTCGTC 3') was used as the reverse primer. P27 mut construct contains a mutated form of the p27 Nef, coding for a full-length protein in which the myristoylation consensus sequence is abolished by the substitution of the glycine residue in second position into alanine. P25 and p27 mut HIV Nef variants were cloned in the binary vector pBI121. To allow transient expression by protoplast transfection, the DNA cassettes starting from the CaMV 35S promoter and ending with the NOS terminator sequence (Figure 1) were cloned into the vector pGEMR-4Z vector for transient expression.
For generation of the p27 construct, the Nef coding sequence was amplified from plasmid pSCNef51 using the P27 Xba1 Forward (5' CAGAGTCGTCTAGAGGTGGCAAGTGGTCAAAAAGT 3') and Nef Flag Reverse (5' 3') primers. The resulting PCR product was cloned into the pDHA vector  for transient expression.
The PR1 signal peptide was amplified from plasmid pDE300d  using the sPR1BamH1Forward (5' CAGAGTCGGGATCCATGGGATTTTTTCTCTTTTC 3') and sPR1Xba1Reverse (5' CAGAGTCGTCTAGACGCATGAGAAGAGTGAG 3') primers. To amplify the sp-Nef variants for cloning into pDHA, the following forward primers were used: p25Xba1For (5' CAGAGTCGTCTAGAAGACGAGCTGAGCCAG 3') and P27Xba1For (5' CAGAGTCGTCTAGAGGTGGCAAGTGGTCAAAAAGT 3'). The NefPst1Rev (5' CAGAGTCGCTGCAGCTAATGGTGATGGTGATG 3') was used as the reverse primer.
The constructs p25 and p27 mut were electroporated into A.tumefaciens strain LBA4404. Leaf discs of Nicotiana tabacum cv Petit Havana SR1, were transformed according to the protocol described elsewhere .
Protoplasts prepared from axenic leaves of tobacco were subjected to polyethylene glycol-mediated transfections exactly as described by . 40 μg of each plasmid was used to transform 106 protoplasts in 1 ml. After transfection, cells were incubated for 16 h at 25°C before metabolic labelling.
In vivo labelling of protoplasts and analysis of expressed polypeptides
Pulse-chase experiments, immunoprecipitation, SDS-PAGE and fluorography were performed as described previously . Immunoprecipitation was performed with anti FLAG antiserum (Sigma, 1:1000) or anti Nef (EVA#3067.4, NIBSC-CFAR MRC) at a 1:1000 dilution.
ELISA detection and quantification of plant-expressed Nef variants
Expression of p25 and p27 mut Nef variants, in tobacco plants, were determined by direct ELISA. TSP were obtained as described: for each sample, 100 mg of leaf tissue was ground thoroughly in liquid nitrogen and the powder was added with 500 μl of PBS-buffer (PBS) containing protease inhibitors (Complete EDTA Free, ROCHE). Extracts were clarified by centrifugation at 20000 × g for 30 min at 4°C. ELISA assays were performed coating 96-well microplates with 100 μl of total protein extract for each sample, for 2 h at 37°C or O/N at 4°C. E.coli recombinant Nef (EVA#650, NIBSC-CFAR MRC) was used as positive control. The plates were blocked with 5% milk/PBS at 37°C for 2 h. After washing, plant recombinant Nef was detected using the HIV-1 Nef rabbit Antiserum (Cat#2949 NIH AIDS Research and Reference Reagent Program)  diluted 1:100 in PBS 2% milk, at 37°C for 2 h or O/N at +4°C, followed by incubation with Biotin-Labeled Affinity Purified anti-rabbit IgG (Cat#16-15-06 KPL) diluted 1:2500 and strepavidin-horseradish peroxidase conjugate (RPN 1231 Amersham) diluted 1:2000, at 37°C for 2 h. The substrate was OPD (AGDIA kit). The reaction was stopped after 1 h, by addition of 1/2 volume of 3 M H2SO4 and the colorimetric reaction was measured at 492 nm. Plant recombinant Nef levels, expressed as% TSP, were estimated using, as standard curve, different concentrations (ranging between 100 and 12.5 ng) of E.coli recombinant Nef (EVA#650, NIBSC-CFAR MRC). The amount of total soluble proteins (TSP) was estimated by Bradford assay. The OD value from each sample was subtracted from control plant OD value.
Immunoblot analysis of expressed polypeptides
Leaf samples (10 mg) were ground in 50 μl of 1× SDS-PAGE sample buffer and centrifuged at 12,000 × g for 10 min. The supernatants were boiled and loaded on 15% SDS-PAGE (w/v) followed by immunoblotting with FLAG antiserum (Sigma, UK). Signal was detected by ECL Plus (Amersham).
Cobalt affinity purification of 6× histidine-tagged Nef
Leaves of a transgenic tobacco plant expressing p27 mut were harvested and ground in liquid nitrogen. The sample was thawed in the presence of lysis buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl) + protease inhibitors (Complete EDTA-free, Roche). The sample was centrifuged at 12,000 × g for 30 minutes at 4°C to pellet the debris. Purification was carried out in a batch method as detailed in the manufacturer's instructions. Briefly, the clarified sample was added to 2 mls of Talon™ cobalt resin (Clontech) sand mixed at room temperature for 30 minutes. The resin was washed twice with 10 column volumes of lysis buffer before being added to a plastic column. The resin was washed with 2 column volumes of wash buffer (lysis buffer + 10 mM imidazole). Proteins were eluted in 1 ml fractions by the addition of elution buffer (lysis buffer + 50 mM imidazole). Fractions were analysed by SDS-PAGE followed by immunoblotting with Flag antiserum (Sigma, UK).
This work was supported by the European Framework VI Integrated Project 'Pharma-Planta'. We are grateful to Dr. K. Krohn, Mikrogen and NIBSC Centralised Facility for AIDS Reagents supported by EU Programme EVA (contract QLK2-CT-1999-00609) and the UK Medical Research Council, for reagent: EVA#650; Dr. M. Harris, Cannon and NIBSC Centralised Facility for AIDS Reagents supported by EU Programme EVA (contract QLK2-CT-1999-00609) and the UK Medical Research Council, for reagent: ARP#2015; Dr. Ronald Swanstrom and NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, for reagent: Cat#2949.
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