cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom

Autores
Clement, Herlinda; Corzo, Gerardo; Neri Castro, Edgar; Arenas, Ivan; Hajos, Silvia Elvira; de Roodt, Adolfo Rafael; Villegas, Elba
Año de publicación
2019
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.
Fil: Clement, Herlinda. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México. Universidad Autónoma del Estado de Morelos; México
Fil: Corzo, Gerardo. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México
Fil: Neri Castro, Edgar. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México
Fil: Arenas, Ivan. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México
Fil: Hajos, Silvia Elvira. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina
Fil: de Roodt, Adolfo Rafael. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Villegas, Elba. Universidad Autónoma del Estado de Morelos; México
Materia
ANTIBODIES
BOTHROPS AMMODYTOIDES
PHOSPHOLIPASE
PROTEIN EXPRESSION
SNAKE
VENOM
VIPER
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/160752
Acceder
id CONICETDig_ac9646feb5d049d5166f4756e7a7dfae
oai_identifier_str oai:ri.conicet.gov.ar:11336/160752
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venomClement, HerlindaCorzo, GerardoNeri Castro, EdgarArenas, IvanHajos, Silvia Elvirade Roodt, Adolfo RafaelVillegas, ElbaANTIBODIESBOTHROPS AMMODYTOIDESPHOSPHOLIPASEPROTEIN EXPRESSIONSNAKEVENOMVIPERhttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.Fil: Clement, Herlinda. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México. Universidad Autónoma del Estado de Morelos; MéxicoFil: Corzo, Gerardo. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Neri Castro, Edgar. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Arenas, Ivan. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Hajos, Silvia Elvira. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: de Roodt, Adolfo Rafael. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Villegas, Elba. Universidad Autónoma del Estado de Morelos; MéxicoAcademic Press Inc Elsevier Science2019-02info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/160752Clement, Herlinda; Corzo, Gerardo; Neri Castro, Edgar; Arenas, Ivan; Hajos, Silvia Elvira; et al.; cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom; Academic Press Inc Elsevier Science; Protein Expression and Purification; 154; 2-2019; 33-431046-5928CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S1046592818301669info:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2018.09.004info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:11:29Zoai:ri.conicet.gov.ar:11336/160752instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 10:11:29.7CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom
title cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom
spellingShingle cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom
Clement, Herlinda
ANTIBODIES
BOTHROPS AMMODYTOIDES
PHOSPHOLIPASE
PROTEIN EXPRESSION
SNAKE
VENOM
VIPER
title_short cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom
title_full cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom
title_fullStr cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom
title_full_unstemmed cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom
title_sort cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom
dc.creator.none.fl_str_mv Clement, Herlinda
Corzo, Gerardo
Neri Castro, Edgar
Arenas, Ivan
Hajos, Silvia Elvira
de Roodt, Adolfo Rafael
Villegas, Elba
author Clement, Herlinda
author_facet Clement, Herlinda
Corzo, Gerardo
Neri Castro, Edgar
Arenas, Ivan
Hajos, Silvia Elvira
de Roodt, Adolfo Rafael
Villegas, Elba
author_role author
author2 Corzo, Gerardo
Neri Castro, Edgar
Arenas, Ivan
Hajos, Silvia Elvira
de Roodt, Adolfo Rafael
Villegas, Elba
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv ANTIBODIES
BOTHROPS AMMODYTOIDES
PHOSPHOLIPASE
PROTEIN EXPRESSION
SNAKE
VENOM
VIPER
topic ANTIBODIES
BOTHROPS AMMODYTOIDES
PHOSPHOLIPASE
PROTEIN EXPRESSION
SNAKE
VENOM
VIPER
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.
Fil: Clement, Herlinda. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México. Universidad Autónoma del Estado de Morelos; México
Fil: Corzo, Gerardo. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México
Fil: Neri Castro, Edgar. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México
Fil: Arenas, Ivan. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México
Fil: Hajos, Silvia Elvira. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina
Fil: de Roodt, Adolfo Rafael. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Villegas, Elba. Universidad Autónoma del Estado de Morelos; México
description A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.
publishDate 2019
dc.date.none.fl_str_mv 2019-02
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/160752
Clement, Herlinda; Corzo, Gerardo; Neri Castro, Edgar; Arenas, Ivan; Hajos, Silvia Elvira; et al.; cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom; Academic Press Inc Elsevier Science; Protein Expression and Purification; 154; 2-2019; 33-43
1046-5928
CONICET Digital
CONICET
url http://hdl.handle.net/11336/160752
identifier_str_mv Clement, Herlinda; Corzo, Gerardo; Neri Castro, Edgar; Arenas, Ivan; Hajos, Silvia Elvira; et al.; cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom; Academic Press Inc Elsevier Science; Protein Expression and Purification; 154; 2-2019; 33-43
1046-5928
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S1046592818301669
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.pep.2018.09.004
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Academic Press Inc Elsevier Science
publisher.none.fl_str_mv Academic Press Inc Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1842270160217440256
score 13.13397

Publicaciones similares