Guidelines to reach high-quality purified recombinant proteins
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Outros Autores: | |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/49312 |
Resumo: | The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity, and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization - denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS), and circular dichroism (CD) - are revisited with focus on the protein, and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design. |
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Guidelines to reach high-quality purified recombinant proteinsRecombinant proteinFusion tagsProtein purificationStructural characterizationQuality controlProtein quantificationScience & TechnologyThe final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity, and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization - denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS), and circular dichroism (CD) - are revisited with focus on the protein, and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.This study was funded by the Fundação para a Ciência e a Tecnologia (FCT), Portugal, under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01- 0145-FEDER-006684) and the Post-Doctoral grant SFRH/BPD/ 110640/2015, and by the BioTecNorte operation (NORTE-01-0145- FEDER-000004) supported by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersionSpringer NatureUniversidade do MinhoOliveira, Carla Cristina Marques deDomingues, Lucília20182018-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/49312engOliveira, Carla; Domingues, Lucília, Guidelines to reach high-quality purified recombinant proteins. Applied Microbiology and Biotechnology, 102(1), 81-92, 20180175-75981432-061410.1007/s00253-017-8623-829151158http://www.springer.com/chemistry/biotechnology/journal/253info: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:RCAAP2023-07-21T12:28:49Zoai:repositorium.sdum.uminho.pt:1822/49312Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:23:42.053364Repositório 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 |
Guidelines to reach high-quality purified recombinant proteins |
title |
Guidelines to reach high-quality purified recombinant proteins |
spellingShingle |
Guidelines to reach high-quality purified recombinant proteins Oliveira, Carla Cristina Marques de Recombinant protein Fusion tags Protein purification Structural characterization Quality control Protein quantification Science & Technology |
title_short |
Guidelines to reach high-quality purified recombinant proteins |
title_full |
Guidelines to reach high-quality purified recombinant proteins |
title_fullStr |
Guidelines to reach high-quality purified recombinant proteins |
title_full_unstemmed |
Guidelines to reach high-quality purified recombinant proteins |
title_sort |
Guidelines to reach high-quality purified recombinant proteins |
author |
Oliveira, Carla Cristina Marques de |
author_facet |
Oliveira, Carla Cristina Marques de Domingues, Lucília |
author_role |
author |
author2 |
Domingues, Lucília |
author2_role |
author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Oliveira, Carla Cristina Marques de Domingues, Lucília |
dc.subject.por.fl_str_mv |
Recombinant protein Fusion tags Protein purification Structural characterization Quality control Protein quantification Science & Technology |
topic |
Recombinant protein Fusion tags Protein purification Structural characterization Quality control Protein quantification Science & Technology |
description |
The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity, and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization - denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS), and circular dichroism (CD) - are revisited with focus on the protein, and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018 2018-01-01T00:00:00Z |
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/1822/49312 |
url |
http://hdl.handle.net/1822/49312 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Oliveira, Carla; Domingues, Lucília, Guidelines to reach high-quality purified recombinant proteins. Applied Microbiology and Biotechnology, 102(1), 81-92, 2018 0175-7598 1432-0614 10.1007/s00253-017-8623-8 29151158 http://www.springer.com/chemistry/biotechnology/journal/253 |
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 |
Springer Nature |
publisher.none.fl_str_mv |
Springer Nature |
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 |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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