Development of a double sandwich ELISA able to discriminate between free PAP (pokeweed antiviral protein) and complexed PAP in leaf extracts

Schlick, Jean-Luc; Desvoyes, Bénédicte; Hakil, M.; Adami, Pascale; Dulieu, Philippe

doi:10.1016/s0168-9452(98)00208-8

Cited by 5 publications

(5 citation statements)

References 27 publications

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“…The cloning strategy was facilitated by the small size of the GnRH peptide (10 amino acids), thus permitting us to directly insert the GnRH sequence at the 5′‐extremity of the PAP cDNA. The choice of the 5′‐extremity of the PAP cDNA was dictated by the known importance of the C‐terminal extremity for PAP cytotoxicity [16,40]. The resulting recombinant toxin GnRH‐PAP was expressed and easily purified to homogeneity by a one step NI 2+ affinity chromatography.…”

Section: Discussionmentioning

confidence: 99%

“…Although the composition of PAPi is still under investigation, hypothetically PAPi may be a self‐protection mechanism of pokeweed cells against PAP which might be misaddressed during its biosynthesis. The amount of PAP versus complexed PAP (in PAPi) can be estimated in a plant extract with an ELISA which discriminates between the two forms [16]. PAP has also been shown to be highly toxic in vitro to cells infected with different animal viruses including the human immunodeficiency virus (HIV) [17–19].…”

Section: Introductionmentioning

confidence: 99%

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Cytotoxic activity of a recombinant GnRH‐PAP fusion toxin on human tumor cell lines

et al. 2000

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Pokeweed antiviral protein (PAP), a ribosomeinactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC 50 of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID 50 of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.z 2000 Federation of European Biochemical Societies.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cytotoxic activity of a recombinant GnRH‐PAP fusion toxin on human tumor cell lines

et al. 2000

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“…2001). Rabbit polyclonal antibodies of PAP were prepared by using purified PAP from the leaves of P. americana that were harvested in spring (Schlick et al. 1999), and monoclonal anti‐PAP antibodies were obtained from mice ascites (Desvoyes et al.…”

Section: Introductionmentioning

confidence: 99%

“…Finally, they have many clinical uses (Chowdhury et al 2001). Rabbit polyclonal antibodies of PAP were prepared by using purified PAP from the leaves of P. americana that were harvested in spring (Schlick et al 1999), and monoclonal anti-PAP antibodies were obtained from mice ascites (Desvoyes et al 1995). Owing to the low expression level of PAP in pokeweed plants, it is complex and time-consuming to extract PAP from pokeweed plants for antibody generation.…”

Section: Introductionmentioning

confidence: 99%

Generation of High Titre Antibodies to Pokeweed Antiviral Protein (PAP) in Rabbits Using Synthetic Peptides and their Use in Detecting PAP in Transgenic Petunia and Yeast

Wang

2012

Journal of Phytopathology

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Antibodies are important for the study of pokeweed antiviral protein (PAP), an important antiviral agent against many plant, animal and human viruses. As PAP is expressed only at a low level in pokeweed plants (Phytolacca americana L.), it is complex and time‐consuming to extract PAP from pokeweed plants for antibody preparation. Here, we describe an antigen‐designed method according to the amino acid sequence that translated from PAP gene cleaving the C‐terminus toxic region and N‐terminus signal peptide (Genbank No. ); the two peptides, DC15: DISGTERQDVETTLC and CR15: CRYPTLESKAGVKSR, were synthesized for generation of antibodies. The design strategy enabled straightforward antigen production and antibody generation. The antibodies can be used to detect PAP in transgenic petunia plants (Petunia hybrida Vilm.), pokeweed plants (P. americana) and transformed yeast (Pachia pastoris), which can express the PAP gene by western blotting. These antibodies generated against synthetic peptides will be useful for various assays such as for PAP detection, immunoprecipitation, protein purification and western blot analysis.

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“…Several investigators have proposed the presence of natural inhibitors for RIPs also (16). The natural inhibitor of a type I RIP from Phytolacca americana, PAP, has been purified and characterized (17,18). However, no such natural inhibitor for the type II RIPs has yet been identified.…”

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confidence: 99%

Crystal Structure of Himalayan Mistletoe Ribosome-inactivating Protein Reveals the Presence of a Natural Inhibitor and a New Functionally Active Sugar-binding Site

Mishra

Bilgrami

Sharma

et al. 2005

Journal of Biological Chemistry

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Ribosome-inactivating proteins (RIPs) are toxins involved in plant defense. How the plant prevents autotoxicity is not yet fully understood. The present study is the first structural evidence of a naturally inhibited form of RIP from a plant. Himalayan mistletoe RIP (HmRIP) was purified from Viscum album leaves and crystallized with lactose. The structure was determined by the molecular replacement method and refined at 2.8-Å resolution. The crystal structure revealed the presence of high quality non-protein electron density at the active site, into which a pteridine derivative (2-amino 4-isopropyl 6-carboxyl pteridine) was modeled. The carboxyl group of the ligand binds strongly with the key active site residue Arg 162 , nullifies the positive charge required for catalysis, and thereby acts as a natural inhibitor. Lectin subunits of RIPs have two active sugarbinding sites present in 1␣-and 2␥-subdomains. A third functionally active site has been identified in the 1␤-subdomain of HmRIP. The 1␤-site is active despite the absence of conserved polar sugar-binding residues. Loss of these residues is compensated by the following: (i) the presence of an extended site where the penultimate sugar also interacts with the protein; (ii) the interactions of galactose with the protein main chain carbonyl and amide nitrogen atoms; (iii) the presence of a well defined pocket encircled by four walls; and (iv) a favorable stacking of the galactose ring with Tyr 66 besides the conserved Phe 75 . The mode of sugar binding is also distinct at the 1␣ and 2␥ sugar-binding sites.

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Development of a double sandwich ELISA able to discriminate between free PAP (pokeweed antiviral protein) and complexed PAP in leaf extracts

Cited by 5 publications

References 27 publications

Cytotoxic activity of a recombinant GnRH‐PAP fusion toxin on human tumor cell lines

Cytotoxic activity of a recombinant GnRH‐PAP fusion toxin on human tumor cell lines

Generation of High Titre Antibodies to Pokeweed Antiviral Protein (PAP) in Rabbits Using Synthetic Peptides and their Use in Detecting PAP in Transgenic Petunia and Yeast

Crystal Structure of Himalayan Mistletoe Ribosome-inactivating Protein Reveals the Presence of a Natural Inhibitor and a New Functionally Active Sugar-binding Site

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