Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris

Detalhes bibliográficos
Autor(a) principal: Oliveira, Carla Cristina Marques de
Data de Publicação: 2008
Outros Autores: Felix, W., Moreira, R. A., Teixeira, J. A., Domingues, Lucília
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/1822/7973
Resumo: Frutalin is an α-d-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZαA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces α-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18–20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS–PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide “T-S-S-N”, which connects α and β chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the α-factor prosequence. Endoglycosidase treatment and SDS–PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-α-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62–64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide “T-S-S-N” does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.
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spelling Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastorisGalactose-binding jacalin-related lectinFrutalinPichia pasto­ris expression systemLinker peptideGlycosylationScience & TechnologyFrutalin is an α-d-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZαA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces α-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18–20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS–PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide “T-S-S-N”, which connects α and β chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the α-factor prosequence. Endoglycosidase treatment and SDS–PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-α-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62–64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide “T-S-S-N” does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.Fundação para a Ciência e a Tecnologia (FCTElsevierUniversidade do MinhoOliveira, Carla Cristina Marques deFelix, W.Moreira, R. A.Teixeira, J. A.Domingues, Lucília2008-082008-08-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/7973eng"Protein Expression and Purification". ISSN 1046-5928. 60:2 (Aug. 2008) 188-193.1046-592810.1016/j.pep.2008.04.00818534865info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-05-11T06:15:31Zoai:repositorium.sdum.uminho.pt:1822/7973Portal AgregadorONGhttps://www.rcaap.pt/oai/openairemluisa.alvim@gmail.comopendoar:71602024-05-11T06:15:31Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris
title Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris
spellingShingle Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris
Oliveira, Carla Cristina Marques de
Galactose-binding jacalin-related lectin
Frutalin
Pichia pasto­ris expression system
Linker peptide
Glycosylation
Science & Technology
title_short Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris
title_full Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris
title_fullStr Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris
title_full_unstemmed Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris
title_sort Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris
author Oliveira, Carla Cristina Marques de
author_facet Oliveira, Carla Cristina Marques de
Felix, W.
Moreira, R. A.
Teixeira, J. A.
Domingues, Lucília
author_role author
author2 Felix, W.
Moreira, R. A.
Teixeira, J. A.
Domingues, Lucília
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Universidade do Minho
dc.contributor.author.fl_str_mv Oliveira, Carla Cristina Marques de
Felix, W.
Moreira, R. A.
Teixeira, J. A.
Domingues, Lucília
dc.subject.por.fl_str_mv Galactose-binding jacalin-related lectin
Frutalin
Pichia pasto­ris expression system
Linker peptide
Glycosylation
Science & Technology
topic Galactose-binding jacalin-related lectin
Frutalin
Pichia pasto­ris expression system
Linker peptide
Glycosylation
Science & Technology
description Frutalin is an α-d-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZαA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces α-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18–20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS–PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide “T-S-S-N”, which connects α and β chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the α-factor prosequence. Endoglycosidase treatment and SDS–PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-α-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62–64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide “T-S-S-N” does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.
publishDate 2008
dc.date.none.fl_str_mv 2008-08
2008-08-01T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/1822/7973
url http://hdl.handle.net/1822/7973
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv "Protein Expression and Purification". ISSN 1046-5928. 60:2 (Aug. 2008) 188-193.
1046-5928
10.1016/j.pep.2008.04.008
18534865
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv mluisa.alvim@gmail.com
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