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

Cheng, Hsin-Pai; Chen, Kuan-Chun; Raja, Joseph A. J.; Li, Jian-Xian; Yeh, Shyi-Dong

doi:10.1016/j.jbiotec.2013.02.002

Cited by 14 publications

(32 citation statements)

References 54 publications

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“…The CE is highly conserved among Asia-type tospoviruses [ 45 ]. When the core nine aa residues (nss) of the CE was deleted or the H113 at the center of the nss was mutated, the MAb does not react with the modified CE [ 45 , 46 ] ( Fig 2 ). Our findings suggest that the CE may be a region that interacts with a host or a viral factor vai its unique feature, similar to its interaction with the NSscon-recognizing MAb [ 45 , 46 ].…”

Section: Discussionmentioning

confidence: 99%

“…A common epitope (CE) in WSMoV NSs protein has been identified as a domain of amino acids (aa) 98–120, denoted as NSscon [ 45 ]. Recently, our laboratory has minimized NSscon sequence to 9 amino acids (aa 109–117), denoted as nss [ 46 ]. This highly conserved common epitope of NSs protein may be responsible for important functions during virus infection, such as RSS function and pathogenicity.…”

Section: Introductionmentioning

confidence: 99%

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Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

et al. 2015

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The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) (109KFTMHNQ117), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif (397IYFL400) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

et al. 2015

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“…The nss-tag, a nine-aa oligopeptide of KFTMHNQIF, was derived from a highly conserved epitope present in NSs proteins of all WSMoV-serogroup and IYSV-serogroup orthotospoviruses [37]. The nss-tag has been successfully used for protein detection in the bacterial pET system and is applicable in transient expression by agroinfiltration in plants followed with co-immunoprecipitation to verify the protein-protein interactions in vitro [38]. Thus, the nss-tag is highly efficient for tagging recombinant proteins in bacterial and plant expression systems.…”

Section: Introductionmentioning

confidence: 99%

“…The six aa residues, 211 KGKEYA 216 , designated as the np sequence, which has the full strength and high sensitivity of the reaction with TNP MAb, were used to test the feasibility of their role as an epitope tag in a bacterial expression system. The green fluorescence protein (GFP), ZYMV CP [38] and the chimeric house dust mite allergen (Dp25) [38] were fused with the np sequence at either the N-or C-terminus. All tagged proteins were expressed effectively in bacteria, and the reaction strength with TNP MAb was similar whether the np sequence was attached at the N-or C-extreme.…”

Section: Introductionmentioning

confidence: 99%

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins

Cheng

Tsai

Hsieh

et al. 2021

IJMS

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The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein–protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein–protein interactions.

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“…The reactivity of tag-fused target proteins with the corresponding tag-specific monoclonal antibodies varies according to immunodetection method, such as Western blotting, immunofluorescence staining, immunoprecipitation, or flow cytometry. His-tag fused target proteins such as Nla 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) have expression problems in bacterial or plant expression systems even though other tags don not have problems in the same expression system ( Cheng et al, 2013 ). Dual tags of MBP with His-tag or Arg-tag used to enhance purification efficiency and solubility of the MBP fusion protein ( Routzahn and Waugh, 2002 ).…”

Section: Introductionmentioning

confidence: 99%

Fine Mutational Analysis of 2B8 and 3H7 Tag Epitopes with Corresponding Specific Monoclonal Antibodies

Kim¹,

Cho²,

Sangsawang³

et al. 2016

Molecules and Cells

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Bacteriophytochromes are phytochrome-like light-sensing photoreceptors that use biliverdin as a chromophore. To study the biochemical properties of the Deinococcus radiodurans bacteriophytochrome (DrBphP) protein, two anti-DrBphP mouse monoclonal antibodies (2B8 and 3H7) were generated. Their specific epitopes were identified in our previous report. We present here fine epitope mapping of these two antibodies by using truncation and substitution of original epitope sequences in order to identify minimized epitope peptides. The previously reported original epitope sequences for 2B8 and 3H7 were truncated from both sides. Our analysis showed that the minimal peptide sequence lengths for 2B8 and 3H7 antibodies were nine amino acids (RDPLPFFPP) and six amino acids (PGEIEE), respectively. We further characterized these peptides in order to investigate their reactivity after single deletion and single substitution of the original peptides. We found that single-substituted 2B8 epitope (RDPLPAFPP) and dual-substituted 3H7 epitope (PGEIAD) showed significantly increased reactivity. These two antibodies with high reactivity for the short modified peptide sequences are valueble for developing new peptide tags for protein research.

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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

Cited by 14 publications

References 54 publications

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins

Fine Mutational Analysis of 2B8 and 3H7 Tag Epitopes with Corresponding Specific Monoclonal Antibodies

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