Expression of functionally active helper component protein of Tobacco etch potyvirus in the yeast Pichia pastoris

Virginia Ruiz-Ferrer, Elisa Goytia, Belén Martínez-García, Dionisio López-Abella and Juan José López-Moya

Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, (CIB, CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain

Correspondence
Juan José López-Moya
jjlopez{at}cib.csic.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Tobacco etch potyvirus (TEV) is transmitted by aphids in a non-persistent manner with the assistance of a virus-encoded protein known as helper component (HC-Pro). To produce a biologically active form of recombinant TEV HC-Pro protein, heterologous expression in the methylotrophic yeast Pichia pastoris was used. A cDNA encoding the TEV HC-Pro region, fused to a Saccharomyces cerevisiae {alpha}-mating factor secretory peptide coding region, was inserted into the P. pastoris genome using a modified version of the pPIC9 vector. The expressed TEV HC-Pro protein was obtained directly from culture medium of recombinant yeast colonies; it was able to interact with TEV particles in a protein overlay binding assay, and also to assist aphid transmission of purified TEV particles to plants using the aphid Myzus persicae as vector. Our results indicate that P. pastoris provides a rapid and low-cost heterologous expression system that can be used to obtain biologically active potyvirus HC-Pro protein for in vitro transmission assays.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Members of the genus Potyvirus, the largest group of plant viruses, are transmitted by aphids in a non-persistent manner. Aphid transmission of potyviruses requires the acquisition of a virus-encoded helper component (HC-Pro) protein in addition to virions (Govier & Kassanis, 1974), a feature shared with other groups of plant viruses (Pirone & Blanc, 1996; Raccah et al., 2001; Froissart et al., 2002). In the case of potyviruses, for successful transmission the aphids must have access to HC-Pro before or at the same time as the virus particles. This suggests that the HC-Pro might form a ‘bridge’ between the virus particles and the aphid mouthparts, where they are retained and can subsequently be inoculated to plants. Among the findings that support this hypothesis, site-directed mutagenesis studies have mapped in HC-Pro two conserved motifs in which modifications have been associated with loss of transmission activity: the first, located in the N-terminal region of HC-Pro, seems to be critical for virus retention in the stylet of the aphid vector (Blanc et al., 1998); the second, located in the C-terminal half of the protein, is probably implicated in binding to the coat protein (CP) of virions (Peng et al., 1998). In addition, an HC-Pro binding domain has been identified at the N-terminal region of the viral CP (Blanc et al., 1997), overlapping with a highly conserved motif that is also essential for the transmission process (Lopez-Moya et al., 1999).

Purification of functionally active HC-Pro protein from infected plant tissue has been a requirement for studying its role during aphid transmission. However, it has been documented that HC-Pro from some potyviruses cannot be purified using standard procedures described with other potyviruses (Sako & Ogata, 1981; Thornbury et al., 1985). For instance, efforts to purify HC-Pro of Tobacco etch virus (TEV) using procedures developed for the purification of HC-Pro of other potyviruses, or modifications thereof, were unsuccessful (Pirone & Thornbury, 1983; Ammar et al., 1994). Taking advantage of the availability of a full-length clone of TEV from which infectious transcripts can be derived (Dolja et al., 1992), an alternative purification procedure involving fusion of a histidine-tag to the 5' terminus of the HC-Pro sequence was developed (Blanc et al., 1999). This system, based on the affinity of histidine (his)-tagged proteins for Ni2+-charged resin, allowed efficient purification from infected tobacco plants of the TEV hisHC-Pro protein, which was functional in aphid transmission experiments. A similar approach was also used in the purification of hisHC-Pro from Lettuce mosaic virus (LMV) (Plisson et al., 2003). Of course, a serious limitation is that tagging procedures can be applied only to viruses from which full-length infectious clones are available. Interestingly, HC-Pro of two other potyviruses, Zucchini yellow mosaic virus (ZYMV) (Kadouri et al., 1998) and Turnip mosaic virus (TuMV) (Wang & Pirone, 1999), was purified using a Ni2+-charged resin without the inclusion of a histidine tag in the viral protein. However, attempts to use this method for unmodified HC-Pro of other viruses, such as Potato virus Y (PVY), Tobacco vein mottling virus (TVMV) (Kadouri et al., 1998) and Plum pox virus (PPV) (our own unpublished observations), were unsuccessful, indicating that this approach cannot be applied to all potyviruses.

Mutational studies in the context of natural potyvirus infection have been limited to viruses with full-length clones from which infectious transcripts can be produced, and also to mutations that do not affect essential roles during the virus life-cycle. In the case of HC-Pro, its involvement in many important functions (Maia et al., 1996; Urcuqui-Inchima et al., 2001) has precluded the systematic use of mutagenesis, for instance to study the aphid transmission process. In the case of TEV, the N-terminal residues of HC-Pro are dispensable for systemic infection, although their deletion affected transmissibility (Dolja et al., 1993). This fact allowed mutagenesis in residues within this region for studying its role during the aphid transmission process (Llave et al., 2002). However, other regions appear to be essential for the virus, and thus a system for expressing active HC-Pro independently of the potyvirus infection would be useful to unravel structural and functional features of the protein. Several attempts to do that with different potyviruses have been reported. On the one hand, transgenic plant systems have been reported to produce HC-Pro protein (Berger et al., 1989; Carrington et al., 1990; Mallory et al., 2001; Ravelonandro et al., 1993; Savenkov & Valkonen, 2001; Mlotshwa et al., 2002). However, these plant-dependent systems needed a considerable time to be implemented and the protein concentration attainable was usually very low. In most cases the expressed HC-Pro remained untested for transmission, or complex purification procedures were needed in order to test its activity (Berger et al., 1989). As an attractive alternative, expression of transmission active PVY HC-Pro was reported to be obtained from plants infected with a recombinant Potato virus X (PVX) vector (Sasaya et al., 2000). Unfortunately, the stability of the insert cannot be guaranteed, a common feature with proteins expressed by viral vectors. Also, the probable role of HC-Pro in suppression of defence mechanisms and synergistic interactions with other viruses (Llave et al., 2000) could make the use of viral vectors difficult for studies involving HC-Pro protein. Expression systems other than plants, such as Escherichia coli or insect cells infected with baculovirus, produced HC-Pro protein that remained inactive when tested in aphid transmission assays (Thornbury et al., 1993).

In the present work, we searched for a heterologous HC-Pro expression system that could yield biologically active protein. We have expressed TEV HC-Pro protein in the methylotrophic yeast Pichia pastoris, and then used the recombinant protein in experiments to test its biological activity, both to bind to TEV virions in vitro, and to assist aphid transmission of purified TEV particles to plants.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus, host plants, bacteria and yeast strains.
Clone pTEV-HCH10, which contains a full-length copy of the TEV genome with a histidine tag fused to the N terminus of HC-Pro (Blanc et al., 1998), was kindly provided by S. Blanc (INRA, St Cristol les Ales, France). Infectious in vitro transcripts from the pTEV-HCH10 clone can be obtained (Blanc et al., 1999) and used for virus propagation in Nicotiana tabacum L. cv. Xanthi nc plants. TEV particles and TEV hisHC-Pro protein were purified as described previously (Murphy et al., 1990; Blanc et al., 1999) from systemically infected plants 2 weeks post-inoculation with pTEV-HCH10 transcripts (Fig. 1). Nicotiana benthamiana Domin. seedlings were used as the test plant for transmission assays (see below).



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Fig. 1. Diagrammatic representation of the TEV genome and constructs used for expression of the HC-Pro protein. (a) The TEV genomic RNA (10 kb) is represented by a thick line between the 5'-linked VPg protein, depicted as a black circle, and the 3' poly(A) tail. The gene products of the viral ORF are represented as boxes with their names above. The HC-Pro is highlighted in black. Below, the full-length clone pTEV-HCH10 (left) and the yeast expression clone pPIC9{alpha}{beta} TEV HC-Pro (right) are shown. As indicated, hisHC-Pro and HC-Pro proteins were derived from each clone after infection of plants and self-cleavage by P1 and HC-Pro proteinases (left), or after expression in the yeast system with STE13 removal of the {alpha}-mating factor secretory peptide (right). (b) Nucleotide and amino acid sequences of the N-terminal regions of the hisHC-Pro and HC-Pro proteins. The relevant nucleotide sequences of the plasmids are indicated with spaces between the codons. The MluI restriction site used for cloning is marked. The amino acids (single-letter code) corresponding to the wild-type TEV HC-Pro are boxed in black, and the extra amino acids fused to the N terminus are underlined. Arrows indicate processing sites.

 
The haploid P. pastoris strain GS115 his4 (Invitrogen) was used for TEV HC-Pro protein expression, and E. coli strain DH5{alpha} was used for DNA manipulations.

Construction of TEV HC-Pro expression vector.
The pPIC9{alpha}{beta} expression vector was constructed from plasmid pPIC9 (Invitrogen) by insertion of a new cloning site with MluI, NotI and EagI as unique restriction sites. Oligonucleotides 5'-TCGAGAAAAGAGAGGCTGAAGCTTACGCGTGC-3' (sense) and 5'-GGCCGCACGCGTAAGCTTCAGCCTCTCTTTTC-3' (antisense) were hybridized, producing overhangs compatible with XhoI and EagI at the 5' and 3' ends respectively. This adaptor was inserted into pPIC9 digested with XhoI and EagI. The resulting new cloning site of pPIC9{alpha}{beta} was confirmed by DNA sequencing.

The coding region of the TEV HC-Pro gene was amplified by PCR with VentR DNA polymerase (New England Biolabs) using plasmid pTEV-HCH10 as template with primers 5'-CTTCTAGACCTTGCTAAAAG-3' (sense) and 5'-GGCCGGCCGTTATCCAACATTGTAAGTTTTCATTTC-3' (antisense), corresponding to positions 843–862, and complementary to 2432–2409 in the TEV genome (Allison et al., 1986). The antisense primer also contained a stop codon and an EagI restriction site. The amplified DNA fragment was digested with MluI and EagI restriction enzymes and cloned into pPIC9{alpha}{beta}. The resulting construct, named pPIC9{alpha}{beta} TEV HC-Pro (Fig. 1), was used to transform E. coli DH5{alpha}, and sequenced to confirm the absence of changes in the HC-Pro sequence, and the correct in-frame fusion of the sequence encoding the {alpha}-mating factor secretory peptide present in pPIC9{alpha}{beta} with the HC-Pro coding region.

Transformation of P. pastoris and selection for positive colonies.
Plasmids pPIC9{alpha}{beta} and pPIC9{alpha}{beta} TEV HC-Pro were digested with either SalI or BglII and used to transform P. pastoris GS115 his4 competent cells in a Bio-Rad GenePulser II apparatus (1500 V charging voltage, 25 µF capacitance, 200 {Omega} resistance). After transformation, cells were plated on Regeneration Glucose Medium (RDB) (1 M sorbitol, 1 % glucose, 1·34 % yeast nitrogen base, 0·00004 % biotin, 0·005 % amino acid mix containing glutamic acid, methionine, lysine, leucine and isoleucine). Recombinant colonies auxotrophic for histidine (His+) were selected by their ability to grow in the absence of this amino acid on plates incubated at 30 °C for 3 days. The phenotype of the transformed P. pastoris colonies was investigated to discriminate between the type of recombination that had occurred: integration at the HIS4 gene with SalI linearized vector yielded a functional AOX1 gene that gave a methanol utilization phenotype (Mut+), while integration/replacement at the AOX1 gene when the vector was linearized with BglII could produce colonies with either a functional AOX1 gene (Mut+) or AOX2 gene (MutS; slow methanol utilization phenotype). Screening for the two phenotypes (His+ Mut+ and His+ MutS) was performed by patching the His+ colonies on replica plates containing minimal glucose (MD) (1·34 % yeast nitrogen base, 0·00004 % biotin, 0·5 % glucose) vs minimal methanol (MM) (1·34 % yeast nitrogen base, 0·00004 % biotin, 0·5 % methanol) plates. Comparison with standard controls albumin GS115 (MutS) and {beta}-Gal GS115 (Mut+), provided by the supplier, were used to identify the methanol-growth phenotype.

Genomic DNA was isolated from P. pastoris as described by the supplier, and the presence of inserts in the P. pastoris genome was checked by PCR. Alternatively, PCR screening was performed directly on lysed P. pastoris cells. Primers used were 5'-GACTGGTTCCAATTGACAAGC-3' and 5'-GCAAATGGCATTCTGACATCC-3' (specific for the AOX1 promoter and terminator sequences, respectively) or the above described TEV HC-Pro specific antisense primer together with primer 5'-CCGTACGTAAGCGACAAATCAATCTCTGAGGCATTC-3' (sense), corresponding to positions 1056–1082 in the TEV genome.

Expression of TEV HC-Pro protein in P. pastoris.
Small-scale expression experiments were performed to test whether the transformed P. pastoris recombinant colonies were suitable for TEV HC-Pro expression. Transformed P. pastoris colonies were cultured for 16–18 h at 30 °C in 5 ml Buffered Glycerol-complex Medium (BMGY) (1 % yeast extract, 2 % peptone, 100 mM potassium phosphate pH 6·0, 1·34 % yeast nitrogen base, 0·00004 % biotin, 1 % glycerol). Cells were collected by centrifugation and resuspended in one-fifth of the original volume of Buffered Methanol-complex Medium (BMMY) (1 % yeast extract, 2 % peptone, 100 mM potassium phosphate pH 6·0, 1·34 % yeast nitrogen base, 0·00004 % biotin, 1 % methanol) for induction of the AOX1 promoter. These cultures were maintained for 4 days (30 °C and 250 r.p.m.) and supplemented daily with methanol to reach a final concentration of 0·5 %.

Selected colonies from the small-scale experiments were chosen for large-scale (700 ml) production of TEV HC-Pro protein. At 24, 48, 72 and 96 h after induction, aliquots (1 ml) of cells were harvested and stored at -80 °C prior to analysis by SDS-PAGE. After 4 days, the culture was centrifuged for 5 min at 3000 g. The supernatant was centrifuged at 90 000 g for 1 h and concentrated by consecutive ultrafiltrations through VivaSpin (Amicon) and Centricon (Pall Corporation) filtration units with exclusion limits of 3000 and 10 000 Da, respectively. The first concentration reduced the volume to approximately 56 ml, and the second one to 0·5 ml. Supernatants from cultures of colonies transformed with pPIC9{alpha}{beta} (empty vector) were used as negative controls for the experiments after following the same induction and concentration steps.

Serological detection of TEV HC-Pro, SDS-PAGE and Western blot.
The production of TEV HC-Pro in the supernatant of the yeast culture medium was analysed by slot-blot using a Bio-Dot apparatus (Bio-Rad). Briefly, aliquots (10 and 100 µl) of supernatants were vacuum-blotted onto PVDF Hybond-P membranes (Amersham). After blocking the membranes with PBST (phosphate-buffered saline with 0·05 % Tween 20) containing 5 % skimmed dry milk powder, they were incubated overnight at 4 °C with the TEV HC-Pro rabbit polyclonal antisera (kindly provided by T. P. Pirone, University of Kentucky, USA) at 1 : 5000 dilution in PBST. After several rinses with PBST and 1 h incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Nordic Immunology) at 1 : 10 000 dilution in PBST, the membranes were rinsed thoroughly with PBST and bound antibodies were detected using the enhanced chemiluminescence ECL system (Amersham).

For Western blot analysis, samples were separated in 4–10 % discontinuous SDS-PAGE following standard methods, and the proteins were transferred to PVDF membranes using a Mini Trans-blot Mini Protean II cell (Bio-Rad) according to the manufacturer's instructions. Detection of TEV HC-Pro protein was performed as described above.

HC-Pro/CP binding assays.
Interactions between TEV HC-Pro and CP from virions were assayed following a previously described protein blotting-overlay technique (Blanc et al., 1998). The HC-Pro protein was applied to Protran nitrocellulose membranes (Schleicher & Schuell) using a vacuum pump. After blocking for 1 h with 5 % skimmed dry milk powder in TSM (100 mM Tris/HCl, pH 7·2, 20 mM MgCl2), the blot was incubated overnight in the same buffer with purified TEV virions at a concentration of 2 µg ml-1. The blots were washed with 5 % skimmed dry milk powder in TSM three times and once in TTBS (100 mM Tris/HCl, pH 7·5, 500 mM NaCl, 0·02 % Tween 20) and incubated overnight with the polyclonal anti-TEV virion antibodies (kindly provided by T. P. Pirone) at dilution 1 : 5000 in TTBS containing 5 % skimmed dry milk powder. After washing for 30 min in the same buffer, the membranes were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (1 : 10 000 dilution) also in TTBS containing 5 % skimmed dry milk powder. After several washes, bound antibodies were detected by using the ECL system.

Aphid transmission assays.
Apterous Myzus persicae Sulzer aphids reared on N. tabacum plants were collected and kept in glass vials for 2–3 h of preacquisition fasting. Groups of starved aphids were allowed to feed during a 10 min acquisition access period through stretched Parafilm membranes on a mixture of purified TEV virions (200 µg ml-1), yeast-expressed TEV HC-Pro protein (100 µg ml-1) and 20 % sucrose prepared in TSM buffer. As positive controls for transmission, TEV hisHC-Pro protein purified from TEV-HCH10-infected plants was also used at similar concentration unless otherwise indicated. Groups of 10 aphids were then placed on N. benthamiana test plants for inoculation and allowed to feed overnight before spraying with insecticide. Symptoms were recorded 10 days later by visual inspection and confirmed by RT-PCR amplification of total nucleic acids extracted from plants using TEV specific primers [5'-CCGTACGCGTCTGGCACTGTGGATGCTGG-3' and 5'-CGGCGGCCGTCACTGGCGGACCCCTAATAG-3', corresponding to positions 8516–8527 (sense) and 9286–9304 (antisense) in the TEV genome, respectively].


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transformation and selection for positive clones
Integration of the TEV HC-Pro gene into the P. pastoris genome was performed by recombination with pPIC9{alpha}{beta} TEV HC-Pro (Fig. 1). Plasmids pPIC9{alpha}{beta} and pPIC9{alpha}{beta} TEV HC-Pro were digested with either SalI or BglII. After digestion the expected products were visualized by agarose gel electrophoresis and used to transform P. pastoris GS115 competent cells. More than 100 colonies of transformed P. pastoris selected by their ability to grow in the absence of histidine (His+) were obtained in all transformation experiments.

Growth rates in the presence of methanol were tested for 100 recombinant His+ colonies from RDB plates: 50 from plates transformed with vectors linearized with SalI, and 50 from plates transformed with vectors linearized with BglII. As expected, all colonies transformed with SalI-linearized vectors exhibited a Mut+ phenotype; in the case of BglII linearized vectors about 20 % of the colonies had a MutS phenotype. No differences in the proportion of growth phenotypes were observed between colonies transformed with empty or TEV HC-Pro containing vectors.

To confirm that the HC-Pro gene had integrated into the P. pastoris genome, genomic DNA from colonies transformed with empty or TEV HC-Pro-containing vectors was analysed by PCR with external or TEV HC-Pro-specific primers. PCR products corresponding to fragments of the expected sizes of the empty vector (484 bp) and the TEV HC-Pro-containing transformants (1375 bp) were observed after agarose gel analysis (Fig. 2a, lanes 1 and 3–6). All colonies transformed with the TEV HC-Pro construct and analysed gave positive results when tested by PCR.



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Fig. 2. Identification of P. pastoris recombinant colonies and expression of the TEV HC-Pro protein. (a) Agarose gel analysis of PCR amplification products using genomic DNA of P. pastoris colonies obtained by recombination with SalI-digested plasmids pPIC9{alpha}{beta} (lane 1) and pPIC9{alpha}{beta} TEV HC-Pro (lanes 3–6). Arrows indicate fragments amplified with external primers (lane 1, 484 bp) or TEV HC-Pro-specific primers (lanes 3–6, 1375 bp). DNA size standards corresponding to bacteriophage {lambda} DNA digested with EcoRI and HindIII were included (lane 2). (b) Slot-blots incubated with anti-TEV HC-Pro polyclonal antibodies after small-scale TEV HC-Pro protein expression with P. pastoris recombinant colonies obtained with SalI- (row 1) or BglII- (row 2) digested pPIC9{alpha}{beta} TEV HC-Pro. Two samples (10 µl and 100 µl) of supernatant were blotted, as indicated. Equal volumes of supernatant of recombinant colonies transformed with SalI- or BglII-digested empty vectors (rows 3 and 4), and two dilutions of plant purified TEV hisHC-Pro protein (row 5) were also included. (c) Western blot detection using anti-TEV HC-Pro polyclonal antibodies of plant purified TEV hisHC-Pro protein (lane 1) and concentrated supernatant of a recombinant colony transformed with SalI-digested pPIC9{alpha}{beta} TEV HC-Pro (lane 2). In addition to the 53 kDa product, the antibody recognized in the plant sample several larger and smaller bands that could represent multimer aggregates and degradation products, respectively. (d) Western blot detection using anti-TEV HC-Pro polyclonal antibodies of a large-scale expression of TEV HC-Pro protein from a recombinant P. pastoris colony transformed with SalI-digested pPIC9{alpha}{beta} TEV HC-Pro. The time-course expression of TEV HC-Pro protein was monitored every 24 h from 0 to 96 h (lanes 3–7) after induction, loading 10 µl of clarified supernatant per well. The two concentrated samples after ultrafiltration were also included (lanes 8 and 9), loading 10 µl and 1 µl of the recovered material after purification with the 3000 and 10 000 Da membranes, respectively. Plant purified TEV hisHC-Pro protein (lane 1) and the supernatant of a recombinant colony transformed with SalI-digested pPIC9{alpha}{beta} empty vector at 96 h after induction (lane 2) were also included.

 
Expression of TEV HC-Pro protein in P. pastoris
Recombinant colonies with the TEV HC-Pro gene integrated in their genomes were used for small-scale expression experiments in BMMY medium. Expression of the viral protein was confirmed by slot-blot serological analysis after blotting 10 and 100 µl of culture media supernatant onto PVDF membranes (Fig. 2b, rows 1 and 2). Several recombinant colonies from MM plates were analysed: not all expressed detectable amounts of TEV HC-Pro protein, although the presence of the insert was confirmed by PCR. The proportion of colonies expressing detectable protein varied among different transformation experiments. Culture media supernatants from colonies transformed with the pPIC9{alpha}{beta} empty vector (Fig. 2b, rows 3 and 4), and TEV hisHC-Pro protein purified from infected tobacco plants (row 5) were also included as controls.

The small-scale experiments showed that both Mut+ and MutS recombinant P. pastoris colonies were able to express the TEV HC-Pro protein. According to the supplier, Mut+ colonies can produce larger amounts of protein in shorter periods of time compared to MutS colonies; therefore Mut+ colonies were chosen for the rest of the study. For that reason, transformation after digestion of plasmid with SalI was preferred because in this case all recombinant colonies presented a Mut+ phenotype.

Large-scale production of TEV HC-Pro protein from recombinant P. pastoris colonies transformed with SalI-digested pPIC9{alpha}{beta} TEV HC-Pro was performed. Western blot analysis showed that the electrophoretic mobility of the P. pastoris-expressed TEV HC-Pro protein was apparently identical to that of TEV hisHC-Pro protein obtained from plants, and no degradation products of the yeast-expressed TEV HC-Pro were detected (Fig. 2c). The time-course of expression was also followed by Western blot analysis. TEV HC-Pro protein was first detected in the culture supernatant at 48 h post-induction, and the amount of protein built up after 72 and 96 h (Fig. 2d, lanes 5–7). The final concentration of the expressed TEV HC-Pro protein was analysed after passing consecutively through 3000 and 10 000 Da ultrafiltration units (Fig. 2d, lanes 8 and 9). Plant purified TEV hisHC-Pro protein and supernatant of a yeast colony transformed with pPIC9{alpha}{beta} empty vector digested by SalI at 96 h after induction were included as positive and negative controls, respectively (Fig. 2d, lanes 1 and 2). Intensities of Western blot bands were scanned with a densitometer and the concentration of the P. pastoris-expressed TEV HC-Pro protein was estimated by comparison with a standard curve obtained with known amounts of plant purified TEV hisHC-Pro protein. Under our conditions, the yield of P. pastoris-expressed TEV HC-Pro protein was about 0·7 mg per litre of yeast culture medium.

Binding assay of TEV HC-Pro protein produced by P. pastoris to TEV virions
To analyse the binding properties between the TEV HC-Pro protein produced by P. pastoris and TEV virions, two dilutions of the protein were slot-blotted in three nitrocellulose membranes (Fig. 3, lanes 6), together with TEV hisHC-Pro protein from infected tobacco plants (lanes 4), TEV purified virions (lanes 2) and negative controls such as virion purification buffer (20 mM Tris/HCl pH 7·5, 1 mM EDTA), hisHC-Pro protein elution buffer (TSM containing 400 mM EGTA buffer) and concentrated supernatant from a yeast colony transformed with pPIC9{alpha}{beta} digested by SalI (lanes 1, 3 and 5, respectively). The binding assay was performed by incubation of the membrane with a solution containing purified TEV virions; meanwhile the other two membranes (Fig. 3a, b) were incubated with TSM buffer. After several washes, membranes were incubated with polyclonal anti-HC-Pro (Fig. 3a) or anti-TEV virion antibodies (Fig. 3b, c). Bound antibodies were detected by successive incubations with horseradish peroxidase-conjugated anti-rabbit IgG and ECL substrate solution. Binding of virions to plant purified TEV hisHC-Pro and yeast-expressed TEV HC-Pro was observed (Fig. 3c, lanes 4 and 6 respectively). Fig. 3(a, b) showed the direct detection of the initially blotted plant purified TEV hisHC-Pro and yeast-expressed TEV HC-Pro (Fig. 3a, lanes 4 and 6 respectively), and of the purified TEV virions (Fig. 3b, lane 2). This experiment demonstrated that the P. pastoris-expressed TEV HC-Pro protein was able to interact with purified virions.



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Fig. 3. Binding between P. pastoris-expressed TEV HC-Pro protein and TEV virions. Purified TEV virions (lane 2), plant purified TEV hisHC-Pro protein (lane 4), concentrated supernatants of P. pastoris cultures of colonies transformed with SalI-digested pPIC9{alpha}{beta} and pPIC9{alpha}{beta}TEV HC-Pro (lanes 5 and 6 respectively) were slot-blotted in nitrocellulose membranes. The upper row of each membrane was loaded with 1/10 of the amount loaded on the lower row. Virion purification (20 mM Tris/HCl pH 7·5, 1 mM EDTA) and TSM (containing 400 mM EGTA) buffers were also included (lanes 1 and 3). Membranes were incubated first with TSM buffer (a and b) or purified TEV virions (c), followed by probing with polyclonal anti-TEV virion antibodies (b and c), or polyclonal anti-TEV HC-Pro antibodies (a). Bound antibodies were detected by successive treatments with horseradish peroxidase-conjugated anti-rabbit antibodies and ECL substrate solution.

 
To quantify binding intensities, a comparison between densitometric measures of virions bound to the plant purified TEV hisHC-Pro (Fig. 3c, lane 4) and to the yeast-expressed TEV HC-Pro (Fig. 3c, lane 6) was performed. The respective loads were normalized in accordance with the direct detection of each protein (Fig. 3a). The quantification established that the plant purified TEV hisHC-Pro bound virions at least two times more strongly than the yeast-expressed TEV HC-Pro.

Aphid transmission with TEV HC-Pro produced in P. pastoris
To determine if the P. pastoris-expressed TEV HC-Pro protein was able to assist aphid transmission, three independent transmission experiments were done using TEV HC-Pro protein from three different P. pastoris expressions and the results compared to those obtained with plant purified TEV hisHC-Pro. Aphids were allowed to feed through membranes on solutions containing purified TEV virions supplemented with the following preparations: (i) TSM buffer, (ii) concentrated supernatant of a P. pastoris colony transformed with the empty vector, (iii) concentrated supernatant of a P. pastoris colony expressing TEV HC-Pro protein or (iv) plant purified TEV hisHC-Pro protein (Table 1). The results showed that the TEV HC-Pro protein present in the P. pastoris concentrated supernatant was active in aphid transmission (6/150, no. of infected plants/no. of plants tested, 4 % transmission). {chi}2 test (or Yates' continuity correction test when the expected values were lower than 5) indicated that significant differences (P<0·05) existed between the transmission with P. pastoris-expressed TEV HC-Pro and the negative controls, and also between transmission with P. pastoris-expressed TEV HC-Pro and TEV hisHC-Pro purified from plants (40 % transmission).


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Table 1. Aphid transmission of purified TEV virions supplemented with P. pastoris-expressed TEV HC-Pro protein

 
Analysis by SDS-PAGE of P. pastoris supernatants containing expressed TEV HC-Pro protein indicated that the expressed TEV HC-Pro protein accounted for the vast majority of the total protein in the medium, although very low levels of other putative yeast-native proteins were also present (data not shown). To determine whether this low background level of proteins present in the TEV HC-Pro preparations, or any other yeast-derived product present in the medium, might be interfering during transmission, a comparison experiment was performed. Aphids were fed on purified TEV virions supplemented with TEV hisHC-Pro from infected tobacco plants (300 µg ml-1), either in plain TSM buffer or mixed with concentrated supernatant obtained from a culture of P. pastoris transformed with the empty vector. The transmission rates were 19/20 (95 %) and 46/49 (94 %) respectively, with no significant differences among them (P>0·05). These results suggested that the different transmission rate obtained with the TEV HC-Pro protein expressed in P. pastoris compared with the plant produced TEV hisHC-Pro was not likely due to interfering components present in the concentrated supernatant.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This study provides the first evidence of production of functionally active potyvirus HC-Pro protein in a eukaryotic P. pastoris expression system. TEV HC-Pro was expressed in P. pastoris shake-flask cultures at a concentration (about 0·7 mg l-1) that is similar to many other proteins expressed in the P. pastoris system. Yields of other proteins in P. pastoris cultures might reach higher values, although important variations have been reported (Cereghino & Cregg, 2000).

The P. pastoris expression system can be used to obtain at least 1 mg of TEV HC-Pro protein from 1·4 l of culture medium. Compared to this output, purification of 1 mg of his-tagged TEV hisHC-Pro protein from plants has been reported to require processing of at least 50 g of TEV-HCH10-infected tobacco plant material (Blanc et al., 1999). Furthermore, protein expression in yeast Mut+ colonies is obtained after 4 days of induction, while at least a 10–20 day post-inoculation period is a requisite for purification of the his-tagged TEV hisHC-Pro from plants (Blanc et al., 1999).

The TEV HC-Pro protein expressed in P. pastoris was able to bind efficiently to TEV virions although the degree of HC-Pro/CP binding was more than two times weaker compared to binding of TEV hisHC-Pro from plants. Hence, differences in the aphid transmission rates obtained with TEV hisHC-Pro from plants (40 %) and TEV HC-Pro from P. pastoris (4 %) could be correlated with the lower binding capability observed in the in vitro interaction with TEV virions. Similar observations were reported earlier with TVMV CP mutants (Blanc et al., 1997). The involvement of HC-Pro/CP interaction in the aphid transmission mechanism seems to be a general feature for members of the genus Potyvirus (Peng et al., 1998; Wang & Pirone, 1999), and its strength measured by in vitro binding assays might turn out to be a good predictor for the efficiency of the process.

However, the low percentage of aphid transmission using TEV HC-Pro protein expressed by P. pastoris might be due to several other reasons. For instance, failure in any of the steps required during expression (i.e. improper processing of the {alpha}-mating factor secretory peptide as shown in Fig. 1b) might result in a final product with extra residues on its N terminus. Although the electrophoretic mobility of the P. pastoris-expressed protein was apparently identical to the TEV hisHC-Pro protein obtained from plants, variability in the N terminus is commonly found when the {alpha}-mating factor secretory peptide is used to express heterologous proteins in P. pastoris (Cereghino & Cregg, 2000), and therefore we cannot rule out that a certain percentage of our protein contained extra residues. In any case, the presence of several extra residues in the N terminus of the TEV hisHC-Pro from plants did not affect its activity.

Another possible reason for the observed low activity of the yeast-expressed TEV HC-Pro could be the stability of the protein. However, this did not seem to be the case because in our conditions no degradation products were detected by Western blot.

Alternatively, improper post-translational modifications of the expressed HC-Pro have been suggested as one possible explanation of the lack of activity of the potyvirus HC-Pro protein in different expression systems (Thornbury et al., 1993), although there is no information regarding which modifications might be essential to preserve functionality. P. pastoris is capable of adding carbohydrate moieties to secreted proteins (Bretthauer & Castellino, 1999). As mentioned above, our results suggest that the putative glycosylation did not alter significantly the SDS-PAGE mobilities between the control TEV hisHC-Pro protein obtained from plants and the TEV HC-Pro expressed in P. pastoris. However, differences in glycosylation occurring in the different organisms might affect the transmission process and further studies will be needed to clarify their hypothetical involvement in the functionality of the TEV HC-Pro protein.

The P. pastoris yeast expression system is being commonly used in biological research because of its well known advantages (Cereghino & Cregg, 2000). P. pastoris grows rapidly on inexpensive media, and the system allows synthesis and examination of different mutant proteins. In addition minimal medium can be modified, for instance to produce radioactively labelled proteins. In our case, the availability of active untagged TEV HC-Pro will make possible studies of its chemical/physical properties, and correlation of these with its ability to function during transmission. Recently, a first approximation to solve the structure of another potyvirus HC-Pro protein has been reported (Plisson et al., 2003), and the P. pastoris heterologous expression system would be adequate to continue such types of studies.

To summarize, our results show that the P. pastoris expression system is a rapid, easy and low cost way to obtain a biologically active potyvirus HC-Pro for functional studies, including examination of the aphid transmission process. The system might serve to identify functional domains and to perform further structural studies, and it might be applicable also to other viruses, and extended to other viral proteins.


   ACKNOWLEDGEMENTS
 
We wish to thank Dr Stephane Blanc for giving us the pTEV-HCH10 clone, Dr Jose Luis Garcia for helping us with the P. pastoris expression system, Antonio Ruiz Mateo for assistance with the statistical analysis, and Dr Cesar Llave for valuable suggestions and critical discussion. This work was supported by grants BIO2000-0914, BIO 2000-1605-C02-02 and AGL2001-2141 from CICYT (Spain), and 07M-0123-2000 and 07M-0072-2002 from Comunidad de Madrid (Spain). V. R.-F. and E. G. are recipients of PNFPI fellowships from MCyT (Spain), and B. M.-G. is the recipient of a post-doctoral contract from the Comunidad de Madrid (Spain).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 7 August 2003; accepted 19 September 2003.