An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems

Journal of Biotechnology 164 (2013) 510–519

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An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems Hao-Wen Cheng a , Kuan-Chun Chen a , Joseph A.J. Raja a,c , Jian-Xian Li a , Shyi-Dong Yeh a,b,c,∗ a

Department of Plant Pathology, National Chung Hsing University, Taichung 40227, Taiwan, ROC Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan, ROC c NCHU-UCD Plant and Food Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan, ROC b

a r t i c l e

i n f o

Article history: Received 16 October 2012 Received in revised form 31 January 2013 Accepted 1 February 2013 Available online 10 February 2013 Keywords: Tospovirus NSs protein Epitope tag Monoclonal antibody

a b s t r a c t NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, “109 KFTMHNQIF117 ”, denoted as “nss”, retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag’s possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and nontagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. © 2013 Elsevier B.V. All rights reserved.

1. Introduction With the modern advances in genomics, proteomics and bioinformatics, many proteins with different functions for therapeutic, diagnostic or industrial purposes have been produced through various recombinant techniques. Different expression hosts such as bacteria (Zerbs et al., 2009), yeast (Cregg et al., 2009), plant virus (Lico et al., 2008), insect (Jarvis, 2009) and mammalian cell lines (De Jesus and Wurm, 2011) have been developed to express recombinant proteins. In most cases of recombinant protein expression, various problems in production, detection, purification or stability of the expressed proteins are to be resolved, and for which using tag(s) for monitoring the recombinant proteins is a common approach. Protein tags are peptide sequences fused with recombinant proteins for various purposes. Moreover, multiple tags are frequently used for stacking benefits such as detection, purification,

∗ Corresponding author at: Department of Plant Pathology, 250 Kuo-Kuang Rd., Taichung, 402 Taiwan, ROC. Tel.: +886 4 22877021; fax: +886 4 22852501. E-mail address: [email protected] (S.-D. Yeh). 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.02.002

solubilization and immobilization of recombinant proteins (Honey et al., 2001; Nilsson et al., 1996; Pryor and Leiting, 1997; Routzahn and Waugh, 2002). Commonly used peptide tags include polyhistidine-tag (histag) (Porath et al., 1975), polyarginine-tag (Smith et al., 1984), FLAG-tag (Hopp et al., 1988), Strep-tag (Schmidt and Skerra, 1993), c-Myc-tag (Kolodziej and Young, 1991), HA-tag (Neill et al., 1997), maltose binding protein (MBP) (Bedouelle and Duplay, 1988), and glutathione-S-transferase (GST) (Smith and Johnson, 1988). It has been shown that peptide tags can interfere with the structure (Goel et al., 2000; Halliwell et al., 2001), biological activity (Chant et al., 2005; Wu and Filutowicz, 1999) and solubility (Woestenenk et al., 2004) of target proteins. Some affinity tags such as MBP, and GST can enhance the solubility of target proteins (Sachdev and Chirgwin, 1999). Epitope tag is a small peptide sequence that can be recognized by a specific monoclonal antibody (MAb) on the same or different protein molecules. Using a small peptide tag is an approach to minimize structural interference. The aforementioned polyhistidine-tag, polyarginine-tag, FLAG-tag, Strep-tag, c-Myc-tag and HA-tag are commonly used as small peptide tags. The HA-tag is

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derived from the human influenza hemagglutinin that is a surface glycoprotein required for infectivity (Neill et al., 1997). The c-Myctag is derived from the c-myc oncoprotein associated with Burkitt’s lymphoma disease (Kolodziej and Young, 1991). The FLAG-tag is an artificially designed peptide consisting of eight amino acids including the enterokinase cleavage site (Hopp et al., 1988). The Strep-tag is also a synthetic tag that contains eight amino acids which specifically bind to streptavidin (Schmidt and Skerra, 1993). The family Bunyaviridae is a large and diverse group of viruses that infect animal and plant hosts (King et al., 2011). Watermelon silver mottle virus (WSMoV), a member of the only plant-infecting genus Tospovirus in this family (King et al., 2011), has enveloped particles containing three single-stranded nucleic acid segments, L, M and S RNAs (Chu et al., 2001). The NSs protein (49.7 kDa) is a nonstructural protein encoded by the viral strand of WSMoV S RNA and it forms inclusion bodies in the cytoplasm of infected plant cells (Kormelink et al., 1991), and functions as a suppressor of post transcriptional gene silencing (PTGS) (Takeda et al., 2002). Epitope mapping using a specific MAb identified a highly conserved region in the NSs protein, denoted as NSscon, covering a continual epitope from amino acid residues 98 to 120. This region was found to be present in NSs proteins of all WSMoV serogroup tospoviruses (Chen et al., 2006). The reaction between the MAb and the NSscon epitope is highly specific and sensitive, suggesting that the epitope NSscon has the potential to be used as a tag for recombinant proteins. In the present study, we explored the utility of the epitope NSscon as a possible tag for protein analyses. Our results established that the NSscon sequence could be trimmed to a minimal length of nine amino acids (“109 KFTMHNQIF117 ”), denoted as “nss”, without losing its detectability. When it was used to tag proteins expressed by bacterial pET system or Agrobacterium transient expression system in plants, detection efficiency and applicability were found to be superior to those of the commonly used his-tag. The nss sequence was also used in a transient expression by agroinfiltration followed by co-immunoprecipitaion to verify the interaction of ZYMV coat protein and helper component-protease in vitro. In addition, the possible efficacy of nss for post-transcriptional gene silencing suppression was not noticed. We conclude that this new epitope tag is highly efficient for tagging recombinant proteins in both bacterial and plant expression systems.

2. Materials and methods 2.1. Construction of recombinant GFP fused with different lengths of NSscon in pET28b The feasibility of NSscon sequence as an epitope tag was tested, and the minimal length of the NSscon sequence recognizable by the NSscon monoclonal antibody (MAb) (Chen et al., 2006) was determined in the bacterial expression system. All the primers used in this part of the study were listed (Supplementary Data) in the part I of Table 1S. The green fluorescent protein (GFP) open reading frame (ORF) containing the NSscon sequence (GFPNSscon), BamHI, KpnI and XhoI sites at its C-extreme was constructed by three successive PCR (35 cycles: 30 s denaturation at 94 ◦ C; 30 s annealing at 55 ◦ C; 2 min synthesis at 72 ◦ C; followed by a 10 min final extension at 72 ◦ C) using the forward primer P-CACC-GFP coupled with three reverse primers, M-NSsconG1-BK, M-NSsconG2 and MNSscon. The GFPNSscon was introduced into pET28b (Novagen, Darmstadt, Germany) via NcoI and XhoI restriction sites to generate pET28-GFPNSscon-his. The progressively deleted NSscon, either from its N- or Cterminus, were constructed by PCR from pET28-GFPNSscon-his. The primers with added Kpn1, Nco1 or XhoI sites for cloning

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purpose were listed in part II of the Table 1S. Individual constructs of GFP fused with different N-terminal deletions (2–12 aa removed) of NSscon were separately cloned into pET28-GFPNSscon-his via KpnI and XmaI sites (Fig. 1A and B); similarly, those with different C-terminal deletions of NSscon (2–6 aa removed) were separately cloned into pET28-GFPNSscon-his via Nco I and XhoI sites (Fig. 1A and B). The GFP fused with dn11c3 (the deletion form NSscon 11 aa deleted from its N-terminal and 3 aa from C-terminal ends), designated “nss” sequence, was constructed by PCR from the pET28-GFP-dn11 using primers P-CACC-GFP and M-NSscon-d3, and then introduced into pET28-GFPNSscon-his by NcoI and XhoI digestion (Fig. 1A and B). All the resulted recombinant plasmids were verified by sequencing. 2.2. Construction of recombinant proteins tagged with NSscon, nss or poly-histidine for expression in prokaryotic or eukaryotic expression system For using SphI and ApaI restriction sites of primers as cloning sites, pET28b was converted into pET28dsa by abolishing the SphI and ApaI sites of the former by site-directed mutagenesis using the QuickChangeTM XL Site-directed mutagenesis Kit (Stratagene, CA, USA). The pETsa-GFP-his carrying GFP ORF with stop codon and pETsa-GFP carrying GFP ORF without stop codon (Fig. 1C) were constructed by PCR amplification of GFP sequence from pET28GFPNSscon-his using the primer pairs, P-GFP-NSA/M-GFP-BKX and P-GFP-NSA/M-GFP-TGA, respectively, and cloned into the mutated pET28dsa (primers listed in part III of Table 1S of Supplementary Data). In order to compare the efficiencies of different tags, GFP was individually tagged with NSscon, nss-tag and his-tag and the sequences were cloned in bacterial expression vector pET28dsa. The GFP ORF fused with NSscon sequence and stop codon at Cterminal end was amplified by PCR using the primers P-GFP-NSA and M-NSsconTGA, and cloned into pETsa-GFP-his via NcoI and XhoI sites to form pETsa-GFP-NSscon (Fig. 1C). The vector pETsaNSscon-GFP (Fig. 1C) carrying GFP ORF with NSscon sequence at its N-extreme was constructed from pET28-GFPNSscon-his by three successive PCRs using three forward primers, P-NSscon1, P-NSscon2 and P-NSscon, coupled with the reverse primer M-GFPTGA-X. The amplified fragment was cloned into pETsa-GFP via NcoI and XhoI sites. pETsa-GFP-nss (Fig. 1C) carrying GFP ORF with nss sequence and a stop codon at its C-extreme was constructed pETsa-GFP by a modified site-directed mutagenesis PCR (Stratagene, CA, USA) using the primers P-Cnss and M-Cnss. The pETsa-nss-GFP or pETsa-his-GFP carrying GFP with the nss sequence or his-tag at the N-extreme was constructed following the design for pETsa-GFP-nss, using primer pairs P-Nnss/M-Nnss and P-Nhis/M-Nhis, respectively.For tagging other proteins with nss-tag, the ORFs of the NIa protease of Papaya ringspot virus W type (WNpro) (Chen et al., 2008), the helper component-protease (HC-Pro) of Zucchini yellow mosaic virus (ZHC) (Lin et al., 2007), a chimeric house dust mite allergen, which combined partial Dp2 (384 bp) (Lynch et al., 1994) and Dp5 (342 bp) (Lin et al., 1994) ORFs (Dp25), and the nucleocapsid protein of Watermelon silver mottle virus (WNP) (Chen et al., 2005) were amplified from respective constructs using specific primer pairs (part IV of Table 1S). These amplified fragments were individually introduced into the pETsa vector via suitable restriction sites (Fig. 1C). Since the above manipulation, which introduced an inadvertent stretch of nucleotide encoding the HC-Pro cleavable amino acid stretch YRVG/G (Carrington et al., 1989), led to unintended post-translational removal of the tags from HC-Pro, pETsa-ZHC-nss or pETsa-ZHC-his were modified by site-directed mutagenesis PCR using the primer pair P-nss-d6/M-ZHCd6 or P-his-d6/M-ZHCd6 to generate pETsa-ZHCd6-nss or pET-ZHCd6-his.

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Fig. 1. Immunodetection of the minimal length of the tospoviral common epitope NSscon for tagging bacteria-expressed GFP. (A) Construction of the full-length NSscon fused at the C-extreme of GFP in pET28 to form pET28-GFPNSscon-his for expression in the bacterial system. GFP also carried a his-tag following the constructed NSscon. T7p indicates the T7 promotor and T7t indicates the T7 terminator. (B) Progressive amino acid deletions of NSscon, either from N terminal or C terminal end, for determining the minimal length for its immunorecognition in Western blotting using NSscon MAb and GFP antiserum. NSscon indicates the full length tospoviral common epitope (Chen et al., 2006). The “dn” or “dc” indicates progressive amino acid deletions from N- or C-terminus of NSscon; the numbers of deleted amino acids are indicated. The GFPs fused with different deletions of NSscon were expressed by pET28 vector in bacteria. (C) Construction of the different tags fused at either N-extreme or C-extreme of GFP with full length NSscon, the minimal length NSscon (nss) or polyhistidine (his) sequence in a modified pETsa vector and detection of tagged GFPs by Western blotting using NSscon MAb, his-tag MAb or GFP antiserum.

From the ZYMV vector p35ZGFPhis, which expresses Cterminally his-tagged GFP in cucurbit plants (Hsu et al., 2004), p35ZGFPnss was generated by site-directed mutagenesis PCR using the primer pair P-ZGn/M-ZGn. To express N-terminally his-tagged GFP, p35ZnssGFP was generated from p35ZGFPhis by two successive site-directed mutagenesis with the primer pairs P-ZnG/M-ZnG that added the nss sequence at the N-extreme of GFP and PZdhis/M-Zdhis that deleted the his-tag from p35ZGFPhis (primers listed in part V of Table 1S). The GFP ORFs in p35ZnssGFP and pZGFPnss were replaced by the ORFs of WSMoV NP (Chen et al., 2005) via SphI/KpnI sites or the chimeric Dp25 ORF via ApaI/BamHI sites to generate p35ZnssWNP, p35ZWNPnss, p35ZnssDp25 and p35ZDp25nss, respectively. In order to monitor the transient expression of nss-tagged recombinant proteins by agroinfiltration, the sequences of ZYMV HC-Pro, WSMoV NP and GFP with the nss-tag (either at N- or

C-extreme) or without tag were released from pETsa vector and cloned into the binary pBA vector (Niu et al., 2006) via NcoI/XhoI sites to generate the pBA-nss-ZHC, pBA-ZHC-nss, pBA-ZHC, pBAnss-WNP, pBA-WNP-nss, pBA-WNP or pBA-GFP. 2.3. Protein expression and detection by Western blotting or indirect ELISA The plasmids for protein expression were transferred into E. coli BL21 cells (Novagen, Darmstadt, Germany) protein expression was performed as described in the manual of pET system (Novagen, Darmstadt, Germany). The plasmids of ZYMV vector were introduced into the systemic host zucchini squash (Cucurbita pepo L. var. Zucchi) by particle bombardment (Hsu et al., 2004). Bacterial protein and ZYMV-infected tissue samples prepared following standard method were separated on a 12% polyacrylamide

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gel containing sodium dodecyl sulfate (Laemmli, 1970). The resolved protein profiles were electro-blotted onto a nitrocellulose membrane using a Bio-Rad transblot apparatus (Trans-Blot Transfer medium, Bio-Rad, Hercules, CA) and the expressed proteins were separately detected using the NSscon monoclonal antibody (MAb) (10,000× dilution) (Chen et al., 2006), GFP antiserum (5000× dilution) (Hsu et al., 2004), PRSV NIa-Pro antiserum (prepared against bacteria-expressed NIa-Pro in our laboratory, unpublished) (5000× dilution), ZYMV HC-Pro antiserum (Wu et al., 2010) (5000× dilution), Dp5 antiserum (Hsu et al., 2004) (5000× dilution) or WSMoV NP MAb (Lin et al., 2005) (10,000× dilution), following standard procedure. The ZYMV-expressed recombinant proteins were also monitored similarly using the same antisera. The signals were quantified relatively to the untagged proteins by Kodak 1D image analysis software (Eastman Kodak, Rochester, NY). We also examined the feasibility of nss-tagged proteins for detection by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA analysis of nss-tagged GFP and WSMoV NP expressed in bacterial and ZYMV viral vector systems were done with the NSscon MAb (Chen et al., 2006), GFP antiserum (Hsu et al., 2004) WSMoV NP MAb (Lin et al., 2005), as described earlier by Yeh and Gonsalves (1984). 2.4. Suppressor activity assay of nss-tagged recombinant proteins by agroinfiltration In order to monitor the transient expression of nss-tagged recombinant proteins in plants by agroinfiltration, the sequences of ZYMV HC-Pro, WSMoV NP and GFP fused with the nss sequence (either at N- or C-extreme) or without tag were released from pETsa vector and cloned into the binary pBA vector (Niu et al., 2006) via NcoI/XhoI sites to generate the pBA-nss-ZHC, pBA-ZHC-nss, pBAZHC, pBA-nss-WNP, pBA-WNP-nss, pBA-WNP or pBA-GFP. These binary vectors were introduced into Agrobacterium tumefaciens ABI strain by eletroporation and cultured in Luria-Bertani medium (LB) with Kanamycin and spectinomycin at 28 ◦ C overnight. The cells were pelleted down and resuspended to an OD600 of 1.0 in 10 mM MgCl2 containing 0.015 mM acentonsyrigone and kept in room temperature for 3 h. Suspensions of A. tumefaciens ABI carrying pBAGFP (a GFP expressor, driven constitutively by a 35 S promoter, prepared in our laboratory), pBAGFi (a 2/3 GFP ORF construct with inverted repeat and used as a silencing inducer, provided by Dr. Shih-Shun Lin, National Taiwan University, Taiwan) were mixed with individual constructed vectors (ratio 1:1:1) and then injected into the lower side of leaves Nicotiana benthamiana Domin plants of 10 cm height stage by a syringe without needle and the plants were kept at 25 ◦ C. The empty vector pBA (Niu et al., 2006) was used as a negative control. The transiently expressed recombinant proteins at 3 dpi were monitored by chemiluminescent Western blotting (Amersham, Bucks, U.K.) using ZYMV HC-Pro antiserum (Wu et al., 2010), WSMoV NP MAb (Lin et al., 2005) or NSscon MAb (Chen et al., 2006) as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Amersham, Bucks, U.K.) or horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Amersham, Bucks, U.K.) as the secondary antibody. The GFP fluorescence was monitored at 3 dpi by a hand-held UV light B-100AP (UVP, CA, USA) and photographed with a digital camera (D7000, Nikon, Japan) with a Cokin P series filter (Cokin, 84 mm, 524 nm, French). 2.5. Co-immunoprecipitation of nss-tagged proteins in vitro We further examined the possibility of the nss-tag to be used for co-immunoprecipitation analysis of interacting proteins. ZYMV CP with or without nss-tag was first cloned into the pETsa vector (Fig. 1C) by PCR with primers P-ZCP and M-ZCP (Part IV of Table 1S).

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E. coli BL21 cells were transformed with the individual plasmids carrying ZYMV CP (pETsa-nss-ZCP, pETsa-ZCP-nss or pETsa-ZCP). ZYMV HC-Pro sequences with or without the nss-tag (pETsa-nssZHC, pETsa-ZHCd6-nss or pETsa-ZHC) were also used for this study. Following induced expression of recombinant proteins, E. coli cells were pelleted and resuspended in 1 ml extraction buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5% Triton X-100, 5% glycerol, 1 mM EDTA and 0.02% NaN3 ) containing protease inhibitor cocktail (Roche Diagnostics, IN, USA), lysed with a sonicator 250–450 Sonifier Analog Cell Disruptor (Branson, CT, USA) and then centrifuged at 13,000 rpm for 5 min. The presence of recombinant proteins in soluble fractions was confirmed with the antiserum against ZYMV CP (Hsu et al., 2004) or HC-Pro (Wu et al., 2010). Aliquots of each 300 ␮l sample containing nss-tagged HC-Pro or nss-tagged CP were mixed with each 100 ␮l sample containing non-tagged CP or nontagged HC-Pro (i.e., nssZHC + ZCP; ZHCnss + ZCP or nssZCP + ZHC; ZCPnss + ZHC) and incubated at 4 ◦ C for 1 h. Purified IgG of NSscon MAb (100 ␮g, described above) was added to each reaction mixture and incubated at 4 ◦ C for 1 h. Then 25 ␮l Mag Protein A Sepharose (GE Healthcare Life Sciences, Uppsala, Sweden) was added and the mixture was incubated further for 1 h. The tubes were kept on a magnetic platform (MagRack6) to capture the Mag Protein A Sepharose beads. After washing with 600 ␮l extraction buffer two times, the beads were resuspended in 100 ␮l sample buffer and the immunoprecipitated proteins were analyzed by Western blotting using the antiserum against ZYMV HC-Pro (Wu et al., 2010) or CP (Hsu et al., 2004) to detect the non-tagged protein pulled down by NSscon MAb through co-immunoprecipitation with the nss-tagged proteins.

3. Results 3.1. Determination and applicability of the minimal sequence of NSscon in prokaryotic protein expression system The NSscon sequence (23 aa; residues 98–120 of NSs protein) was used for tagging GFP expressed by the pET28b vector in bacteria (Fig. 1A and B). The GFP-NSscon containing both the NSscon sequence and his-tag was readily detected by Western blotting using NSscon MAb or GFP antiserum (Fig. 1B). GFP tagged with different N-terminal deletions of NSscon (Fig. 1B) were expressed and monitored by Western blotting using NSscon MAb. The NSscon MAb readily recognized the deletion forms of NSscon with up to 11 aa removed from the N-terminal end, but failed to recognize the 12 aa-deleted NSscon (Fig. 1B). Our results established that the amino acid residues upstream of K109 of NSscon are dispensable for recognition by the MAb, whereas residue K109 is indispensable. The MAb also recognized the NSscon sequences having 2, 3 and 4 residues deleted from the C-terminal end, but failed to recognize the peptide when 5 or 6 residues were removed (Fig. 1B). These results indicated that residue F117 at the C-terminal region of NSscon is important for MAb recognition, as the reactivity was greatly diminished upon its deletion. Thus, the nine amino acid stretch “109 KFTMHNQIF117 ” of the NSscon (Fig. 1B) is the minimal length for its recognition by the NSscon MAb; this minimal peptide was designated as “nss”. Serial constructs were generated on a modified pETsa vector to express NSscon-, nss- or his-tagged GFP in a bacterial expression system to compare the detectability of different tags (Fig. 1C). The recombinant GFPs carrying the different tags, either at the Nor C-terminus were monitored by Western blotting using NSscon MAb (Chen et al., 2006), his-tag MAb (Invitrogen, Carlsbad, USA) or GFP antiserum (Hsu et al., 2004). Expression of the recombinant GFPs was verified by GFP antiserum and quantified relatively to the untagged GFP. The expression level of GFP tagged with

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either the whole NSscon or the minimal nss at the N-extreme was higher than that for untagged GFP (Fig. 1C). However, expression levels were lower when either of these tags was individually fused at the C-terminus of GFP; this was especially noticed for the GFP-NSscon fusion (Fig. 1C). Importantly, the expression levels of NSscon- or nss-tagged GFP were comparable to those of his-tagged GFP, indicating that both NSscon and nss can efficiently tag GFP for its expression in the bacterial system. Also, the reaction strength appeared to be similar for NSscon and nss, regardless of their fusion at the N- or C-terminus of GFP.

3.2. Application of the nss-tag in the bacterial and plant viral expression system In addition to GFP, the NIa protease of PRSV-W (WNpro), the HC-Pro of ZYMV (ZHC), the nucleocapsid protein of WSMoV (WNP) and a chimeric house dust mite allergen (Dp25) were chosen to further test the feasibility of the nss sequence for tagging different recombinant proteins expressed by bacterial pETsa system. Our results established that the four test proteins fused with the nss sequence, either at their N- or C-terminus, were readily

Fig. 2. Application of the nss-tag in the bacterial and Zucchini yellow mosaic virus vector expression system. (A) Various proteins were expressed in the bacterial vector system, including NIa protease of Papaya ringspot virus W type (WNpro), HC-Pro of Zucchini yellow mosaic virus (ZHC), house dust mite chimeric allergen (Dp25) and nucleocapsid protein of Watermelon silver mottle virus (WNP). (B) The expression levels of GFP, WNP and Dp25 proteins expressed from ZYMV vector system were determined by Western blotting. Individual proteins fused with the nss-tag or his-tag, either at their N- or C-extremes, were detected by NSscon MAb, his MAb, PRSV NIa-Pro antiserum, ZYMV HC-Pro antiserum, Dp5 antiserum or WSMoV NP MAb. The untagged recombinant proteins were used as controls. NI indicates the non-induction bacterial fraction. To avoid the self-proteolysis of HC-Pro, the six aa of HC-Pro cleavage site (YRVG/G) inadvertently introduced at its C-extreme was removed to retain the tag sequence (ZHCd6nss and ZHCd6his).

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Fig. 3. Detection of bacterially expressed (A) and Zucchini yellow mosaic virus vector-expressed (B) nss-tagged GFP and nss-tagged WSMoV NP by indirect ELISA using NSscon MAb. The “Bacteria” indicates the bacteria-expressed recombinant proteins and the “ZYMV” indicates the recombinant proteins expressed by Zucchini yellow mosaic virus vector in squash plants. The histogram bars with oblique lines represent the detection by NSscon MAb, the bars with black dots represent detection by GFP antiserum, and the bars with small squares represent detection by Watermelon silver mottle virus (WSMoV) NP MAb. The statistical analysis of the reaction of NSscon MAb was performed by ANOVA.

detected by the NSscon MAb, except for the HC-Pro-nss fusion (Fig. 2A). This isolated failure was understood to be because of a HC-Pro cleavage site (YRVG/G) inadvertently introduced during the construction, which allowed an autocatalytic cleavage by HC-Pro (Carrington et al., 1989) that removed the tag following translation. Restoration of detection of nss-tag was accomplished by deletion of the cleavage site from the C-terminal region of HC-Pro (Fig. 2A, ZHCd6nss); however, the his-tag was still not detected (Fig. 3B, ZHCd6his). Expression levels of all recombinant proteins were similar, as shown by Western blot assays using the individual antibodies against each protein (Fig. 2A). WNpro and WNP with his-tag at their N-termini and ZHC-Pro with his-tag at its C-terminus were not detected, while his-tagged Dp25 at its N-terminus was barely detected. When an equal amount of 5 ␮g purified IgG was used for comparison, the nss-tagged GPFs were detected at 256× antigen dilution by NSscon MAb, comparable to the detection sensitivity of the protein by GFP antiserum but superior to the dectectablility of the his MAb which only detected his-tagged GFP up to 32× dilution (Fig. 1S). These results indicated that the nss-tag has greater detection sensitivity for recombinant proteins than the his-tag. The nss sequence was further tested for expression of tagged recombinant proteins using a ZYMV vector in squash plants (Fig. 2B). The nss sequence was fused at N- and C-termini of GFP,

WNP and Dp25 and expressed by ZYMV vector. All the recombinant viruses were infectious and the nss-tagged recombinant proteins were detected by NSscon MAb by Western blotting from squash plants (Figs. 2B and 3B). The nss-tagged GFP and WNP expressed by bacterial or ZYMV vector system were also readily detected by ELISA using NSscon MAb (Fig. 3) Overall, our results indicate that the recombinant proteins tagged with the nss sequence can be readily detected by the NSscon MAb, regardless of the nss-tagged terminus and that the nss sequence is suitable to be used as an epitope tag in both bacterial and plant viral vector expression system. 3.3. The nss-tag does not interfere with functions of tagged proteins Since the nss sequence is derived from the common epitope of the tospoviral PTGS suppressor, NSs protein (Takeda et al., 2002), we further examined the possibility for nss-tag’s functional interference with the tagged ZYMV HC-Pro, a gene silencing suppressor, and WSMoV NP, a non-gene silencing suppressor. The nss-tagged and non-tagged ZYMV HC-Pro and WSMoV NP were separately constructed in a binary vector and expressed in leaves of N. benthamiana by agroinfiltration. At 3 dpi, recombinant ZYMV HC-Pro tagged with the nss sequence, either at the N- or C-terminus, was

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Fig. 4. Detection and functional assay of transiently expressed Zucchini yellow mosaic virus helper component protease (ZHC, a RNA silencing suppressor) and Watermelon silver mottle virus nucleocapsid protein (WNP, a non-RNA silencing suppressor) tagged with the nss sequence, after agroinfiltration on tobacco plants. (A) Detection of recombinant ZHC and WNP proteins in the infiltrated leaf tissue. The tagged ZHCs or WNPs were detected by Western blotting using HC-Pro antiserum, WSMoV NP MAb or NSscon MAb. (B) Suppressor activity assay of ZYMV HC-Pro and WSMoV NP fused with the nss-tag. The GFP expression construct and GFi, a silencing inducer containing inverted repeat of GFP coding sequence, were coinfiltrated with nss-tagged HC-Pro (a silencing suppressor) or NP proteins (a non-silencing suppressor) on leaf tissues of Nicotiana bethamiana to test the suppression of GFP silencing. The photographs were taken under white light or UV light at 3 dpi. EV indicates the empty vector.

readily detected by HC-Pro antiserum and NSscon MAb (Fig. 4A). The nss-tagged forms of WSMoV NP were expressed to similar levels, based on their detection by the NP MAb and NSscon MAb (Fig. 4A). At 3 dpi, GFP expression was completely silenced when coinfiltrated with the GFi silencing inducer (Fig. 4B). In contrast, fluorescent signals of GFP were similar if coinfiltrated with the GFi construct with either the nss-tagged or non-tagged HC-Pro (Fig. 4B). However, GFP expression was not restored if coinfiltrated with the GFi construct with the nss-tagged or non-tagged NP (Fig. 4B). Our results indicated that nss-tag renders the tagged transiently expressed recombinant proteins immunodetectable and the tag neither interferes with the suppressor activity of ZYMV HC-Pro,

nor confers the function of gene silencing suppression on WSMoV NP. 3.4. Co-immunoprecipitation of nss-tagged ZYMV CP or HC-Pro in vitro The interaction of potyviral coat protein (CP) and HC-Pro is essential for the aphid transmission of potyviruses (Atreya and Pirone, 1993; Granier et al., 1993). In order to test if the nss sequence can be used for co-immunoprecipition analysis of interacting proteins, either the bacteria-expressed nss-tagged ZYMV CP or nss-tagged HC-Pro was treated with the other non-tagged

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Fig. 5. Co-immunoprecipitation of nss-tagged Zucchini yellow mosaic virus CP (A) or nss-tagged HC-Pro (B) using NSscon MAb in vitro. The recombinant ZYMV CP or HC-Pro were expressed in nss-tagged or untagged form by bacterial system. “Input” indicates that the solution contained only the nss-tagged CP or nss-tagged HC-Pro. “IP” indicates that the NSscon MAb was used to pull down the nss-tagged recombinant proteins from the CP/HC-Pro mixed solutions. The presence of nss-tagged or untagged ZYMV CP and HC-Pro from the “Input” and “IP” fractions was detected by Western blotting using ZYMV CP antiserum (As-ZCP) and HC-Pro antiserum (As-ZHC), respectively.

interacting protein and the complex was immunoprecipitated using NSscon MAb. Our results showed that the non-tagged HCPro was co-immunoprecipitated with the nss-tagged CPs (either nssZCP or ZCPnss) by NSscon MAb and was able to be detected by HC-Pro antiserum (Fig. 5A). Similarly, the non-tagged CP was co-immunoprecipitated with nss-tagged HC-Pros (Fig. 5B). Taken together, our results demonstrated the co-immunoprecipitation of non-tagged HC-Pro/CP with nss-tagged CP/HC-Pro, implying that the nss-tag is applicable for co-immunoprecipitation analyses for protein–protein interactions. 4. Discussion Affinity tags are important for detection of recombinant proteins, because it is time-consuming to produce specific antibodies for individual proteins. However, since the affinity tags have the potential to interfere with the structural and functional aspects of the fusion proteins, it is generally desirable that they be removed, or are sufficiently small in order to minimize any adverse effects. Earlier, we identified a 23 aa common epitope, designated NSscon, in the NSs proteins of the WSMoV serogroup of tospoviruses (Chen et al., 2006). In the present study, we used the identified core 9 aa of NSscon, the “nss” sequence of KFTMHNQIF, as an epitope tag for

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labeling various recombinant proteins expressed in different systems of bacteria, plant viral vector and agroinfiltration. Our results indicated that this novel nss sequence is a highly valuable epitope tag for recombinant proteins, as reflected in the superior detection sensitivity as analyzed by Western blotting, indirect ELISA and co-immunoprecipitation using NSscon MAb. Deletion studies of NSscon allowed us to identify a minimal 9 amino acid residues 109 KFTMHNQIF117 (nss) within the NSscon epitope, wherein the removal of the K109 (lysine) and F117 (phenylalanine) totally abolished the immunodetectability of nss by NSscon MAb. Lysine is a positively charged amino acid involved in many post-translational modifications, such as acetylation, methylation, ubiquitination, sumoylation, neddylation, biotinylation and carboxylation (Sadoul et al., 2008). Lysine is also frequently paired with a negatively charged amino acid to stabilize hydrogen bonds that is important for protein stability (Betts and Russell, 2003). Phenylalanine is an aromatic hydrophobic amino acid that is often buried within the hydrophobic core of the protein and it can also involve in stacking interactions with other aromatic amino acid side chains (Betts and Russell, 2003). Thus, deletion of K109 and F117 residues from the nss could well result in a drastic alteration in conformation of the epitope, thereby leading to its failure to be recognized by NSscon MAb. The his-tag is well known to decrease the solubility of the tagged proteins, especially if they are fused with the C-termini of proteins (Woestenenk et al., 2004). Several studies favored the use of solubilizing fusion tags, rather than the his-tag, to maintain/enhance the solubility of target proteins (Braun et al., 2002; Hammarstrom et al., 2002; Shih et al., 2002). Other common drawbacks of the histag are its poorer and/or variable detection sensitivity (Debeljak et al., 2006) and its N- or C-terminal context-dependent differential effect on recombinant protein solubility (Woestenenk et al., 2004). We also encountered several of these difficulties, when we used his-tag in our earlier studies. The nss-tag can be placed at either the N- or C-terminus of GFP and other proteins and detected from either terminus with less position effect than that for the his-tag (Figs. 2 and 3). As shown for GFP expressed in bacterial system (Figs. 1C and 2A) and ZHC and WNP expressed by transient expression system in plant (Fig. 4A), the nss-tagging at the N-termini of recombinant proteins can enhance the cellular levels of the proteins. Sequence elements at termini of proteins can influence their rate of degradation (Flynn et al., 2003; Hayes et al., 2002; Varshavsky, 1997), and hence, some affinity tags may increase the level of recombinant proteins by decreasing their intracellular proteolysis (De Marco et al., 2004). Therefore, the enhanced expression levels by the N-terminally nsstagged proteins may be due to the positive influence on the stability or solubility of the labeled proteins. The his-tag is one of the most widely used affinity tags. It is small and can be placed, as an affinity tag, either at the N- or C-termini of recombinant proteins. The metal ion-coordinating property of the his-tag enables purification of the his-tagged target proteins from crude extracts, in a single step by metal–chelate affinity chromatography using a nickel–nitriloacetic acid (Ni–NTA) resin (Porath et al., 1975). However, there is no single tag that is suitable for every recombinant protein, therefore, combinations of different tags provide useful benefits for recombinant proteins (Waugh, 2005). Applications for multiple affinity tags include detection, purification (Honey et al., 2001; Nilsson et al., 1996), solubilization and immobilization of recombinant proteins (Pryor and Leiting, 1997; Routzahn and Waugh, 2002). As indicated by our results (Fig. 2) the nss-tag’s detection sensitivity may not be compromised, when the nss-tagged proteins are additionally his-tagged. Hence, recombinant proteins can be engineered with both nss-tag and histag for specific immunodetectability by NSscon MAb and efficient purification by Ni–NTA affinity column chromatography.

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Strong and specific immunoreaction of nss-tagged recombinant proteins is corroborative of our earlier observations that the NSs MAb specifically detected the Asia type tospoviruses on tobacco plants and the ZYMV-expressed NSs protein in squash plants (Chen et al., 2006). Moreover, both in Western blotting analyses and ELISA, the NSs MAb did not show any nonspecific cross reactions with host plants of the families Solanaceae and Cucurbitaceae tested so far (Figs. 2B and 3B and unpublished data). Similarly, the bacteria-expressed or plant viral vector-expressed nss-tagged GFPs or WSMoV NPs can also be detected without background with NSscon MAb by indirect ELISA (Fig. 3B). Overall, these results indicate that nss-tag can serve as an efficient detection tag to monitor recombinant proteins both in bacterial and plant systems by ELISA and Western blotting analysis without the interference from cross reactions with host proteins. In this study, we showed that the nss sequence can serve as a highly effective epitope tag for recombinant protein expression. The nss sequence was derived from the common epitope of a tospoviral NSs protein that functions as a suppressor of gene silencing (Takeda et al., 2002). This highly conserved epitope may be responsible for PTGS function and may carry over that effect to the tagged proteins and interfere with their functions. However, the results of our gene silencing suppression assay by agroinfiltration indicated that the nss sequence does not carry over the silencing suppression function of the NSs protein to tagged proteins, ruling out the possibility for interference of nss-tag with the function of tagged proteins. Co-immunoprecipitation is a commonly used approach to analyze protein–protein interactions. The conserved DAG motif of potyviral CP interacts with the PTK motif of potyviral HC-Pro (Blanc et al., 1997; Peng et al., 1998), and this interaction is essential for aphid transmission of potyvirus (Atreya and Pirone, 1993; Granier et al., 1993). Being a tiny peptide of 9 aa, nsstag may not cause significant conformational changes in tagged proteins; however, it retains sufficient affinity to strongly bind to NSscon MAb for co-immunoprecipitation of nss-tagged protein and non-tagged interacting protein(s), as reflected in the co-immunoprecipitation of ZYMV CP and HC-Pro, when either one of the two was nss-tagged. Furthermore, the complex of the bacteria-expressed nss-tagged ZYMV CP and HC-Pro immunocaptured by NSscon MAb bound to protein A-coupled magnetic beads (Fig. 5), revealing the capacity of the nss-tag to serve as a protein purification tag in a protein A-based protein purification system. The hemagglutinin (HA) peptide (YPYDVPDYA) has become a popular epitope tag for detection and immunoprecipitation (IP), because of the strong interaction between HA and HA MAb (Kaboord and Perr, 2008; Kolodziej and Young, 1991). Both HA and nss sequences are derived from viral genes and they are of similar size, suggesting the possibility that the nss-tag has potential to become an effective epitope tag based on the very strong interaction between the nss-tag and NSscon MAb. An independent laboratory in UC Davis, which used the nss-tag system for coimmunoprecipitation of interacting phloem proteins in the phloem system of curcubits found that a single copy nss sequence tagging was adequate to pull down protein complexes, unlike certain other tag systems including HA and c-Myc that required multiple copy tagging (William Lucas, personal communication). Moreover, since the nss-tag does not lose its immunodetectability in a protein context-dependent manner or it does not appear to negatively affect the cellular expression levels of the tagged proteins (Fig. 5), this-tag is promising for immunoprecipitation of cellular and/or pathogenic proteins interacting with the target proteins, if the immunopreciptation conditions for interacting proteins are optimized.

Acknowledgements We thank the supports from National Science Council (NSC 98-2321-B-005-008-MY3, NSC 101-2321-B-005-010 and NSC 1012911-1-005-301) and this work is supported in part by the Ministry of Education, Taiwan, ROC, under the ATU plan. We also thank Dr. Ching-Hsing Hsu who provided us the chimeric house dust mite allergen cDNA clone and Dr. Shih-Shun Lin who provided us the pBAGFi construct. Our gratitude is also extended to Dr. William Lucas for his reviewing of the manuscript and critical suggestions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec. 2013.02.002.

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