Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield
Autor(a) principal: | |
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Data de Publicação: | 2012 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | https://doi.org/10.1002/btpr.1621 http://www.locus.ufv.br/handle/123456789/21779 |
Resumo: | Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min−1, and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L−1). These parameters yielded 4.0 g L−1 of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L−1 of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression. |
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Silveira, Wendel B. daDiniz, Raphael H. S.Passos, Flavia M. L.Casteluber, Marisa C. F.Damasceno, Leonardo M.Passos, Frederico J. V.2018-09-12T16:48:26Z2018-09-12T16:48:26Z2012-08-221520-6033https://doi.org/10.1002/btpr.1621http://www.locus.ufv.br/handle/123456789/21779Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min−1, and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L−1). These parameters yielded 4.0 g L−1 of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L−1 of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression.engBiotechnology ProgressVolume 28, Issue 6, Pages 1419-1425, November/December 2012Pichia pastorisStreptadivinHeterologous protein expressionRecycling of biomassAerationCloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yieldinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALartigo.pdfartigo.pdfTexto completoapplication/pdf295236https://locus.ufv.br//bitstream/123456789/21779/1/artigo.pdf49a77c8ce3f49b16862b29587e10bd10MD51LICENSElicense.txtlicense.txttext/plain; charset=utf-81748https://locus.ufv.br//bitstream/123456789/21779/2/license.txt8a4605be74aa9ea9d79846c1fba20a33MD52THUMBNAILartigo.pdf.jpgartigo.pdf.jpgIM Thumbnailimage/jpeg4966https://locus.ufv.br//bitstream/123456789/21779/3/artigo.pdf.jpg761ea4966cdcf034efe59281b75493e6MD53123456789/217792018-09-12 23:00:41.927oai:locus.ufv.br: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Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452018-09-13T02:00:41LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.en.fl_str_mv |
Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield |
title |
Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield |
spellingShingle |
Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield Silveira, Wendel B. da Pichia pastoris Streptadivin Heterologous protein expression Recycling of biomass Aeration |
title_short |
Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield |
title_full |
Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield |
title_fullStr |
Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield |
title_full_unstemmed |
Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield |
title_sort |
Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield |
author |
Silveira, Wendel B. da |
author_facet |
Silveira, Wendel B. da Diniz, Raphael H. S. Passos, Flavia M. L. Casteluber, Marisa C. F. Damasceno, Leonardo M. Passos, Frederico J. V. |
author_role |
author |
author2 |
Diniz, Raphael H. S. Passos, Flavia M. L. Casteluber, Marisa C. F. Damasceno, Leonardo M. Passos, Frederico J. V. |
author2_role |
author author author author author |
dc.contributor.author.fl_str_mv |
Silveira, Wendel B. da Diniz, Raphael H. S. Passos, Flavia M. L. Casteluber, Marisa C. F. Damasceno, Leonardo M. Passos, Frederico J. V. |
dc.subject.pt-BR.fl_str_mv |
Pichia pastoris Streptadivin Heterologous protein expression Recycling of biomass Aeration |
topic |
Pichia pastoris Streptadivin Heterologous protein expression Recycling of biomass Aeration |
description |
Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min−1, and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L−1). These parameters yielded 4.0 g L−1 of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L−1 of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression. |
publishDate |
2012 |
dc.date.issued.fl_str_mv |
2012-08-22 |
dc.date.accessioned.fl_str_mv |
2018-09-12T16:48:26Z |
dc.date.available.fl_str_mv |
2018-09-12T16:48:26Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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publishedVersion |
dc.identifier.uri.fl_str_mv |
https://doi.org/10.1002/btpr.1621 http://www.locus.ufv.br/handle/123456789/21779 |
dc.identifier.issn.none.fl_str_mv |
1520-6033 |
identifier_str_mv |
1520-6033 |
url |
https://doi.org/10.1002/btpr.1621 http://www.locus.ufv.br/handle/123456789/21779 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartofseries.pt-BR.fl_str_mv |
Volume 28, Issue 6, Pages 1419-1425, November/December 2012 |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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Biotechnology Progress |
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Biotechnology Progress |
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