Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia

Detalhes bibliográficos
Autor(a) principal: de Melo Silva, Alex Jose José
Data de Publicação: 2016
Outros Autores: Alves, Lumar Lucena, Pakay, Julian
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Revista de Ensino de Bioquímica
Texto Completo: http://bioquimica.org.br/revista/ojs/index.php/REB/article/view/638
Resumo: This work was performed as an integrated practical of a Biomedical Science undergraduate course of Biochemistry subject, in order to demonstrate used techniques to purify of Green Fluorescent Protein (GFP). To perform the experiments the main methodology applied was the by immobilized metal affinity chromatography (IMAC).  The open reading frame for enhanced GFP was sub-cloned into the pQE30 expression vector. The subsequent production of protein tagged N-terminally with hexahistidine, facilitated its purification by IMAC.  An approximate 3-fold purification of GFP was achieved. Thus, the students who completed the course gained significant experience related to fundamental techniques in molecular cloning and a sound basis in the principles of recombinant protein expression and purification.
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spelling Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – AustraliaPurificação de Proteína verde fluorescente por meio de Cromatografia de Afinidade com Íon Metal Imobilizado (IMAC) e sua relevância para estudantes de Ciências Biomédicas durante aulas de Bioquímica na Universidade La Trobe – Austráliapractical class, purificationGFP; Purification; IMACAulas práticasGFP; Purificação; IMACThis work was performed as an integrated practical of a Biomedical Science undergraduate course of Biochemistry subject, in order to demonstrate used techniques to purify of Green Fluorescent Protein (GFP). To perform the experiments the main methodology applied was the by immobilized metal affinity chromatography (IMAC).  The open reading frame for enhanced GFP was sub-cloned into the pQE30 expression vector. The subsequent production of protein tagged N-terminally with hexahistidine, facilitated its purification by IMAC.  An approximate 3-fold purification of GFP was achieved. Thus, the students who completed the course gained significant experience related to fundamental techniques in molecular cloning and a sound basis in the principles of recombinant protein expression and purification.Este trabalho foi realizado como prática integrada da disciplina de Bioquímica no curso de graduação em Ciências Biomédicas, objetivando demonstrar técnicas utilizadas para a purificação da Proteina Fluorescente Verde (GFP).  Para realização dos experimentos foi utilizada como metodologia principal a Cromatografia de Afinidade por Metal Imobilizado (IMAC). A região de abertura para alongamento da GFP foi subclonada dentro de um vetor de expressão chamado pQE30. A produção subsequente da proteína marcada com hexahistidina na região N-terminal, facilitou sua purificação pela IMAC. Uma purificação de aproximadamente três vezes mais que a esperada da GFP foi obtida. Dessa forma, os estudantes completaram o curso adquirindo significante experiência em relação às técnicas fundamentais na área molecular relacionadas à clonagem e princípios de expressão e purificação de proteínas recombinantes.Sociedade Brasileira de Bioquímica e Biologia Molecular - SBBqde Melo Silva, Alex Jose JoséAlves, Lumar LucenaPakay, Julian2016-12-15info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionPracticalapplication/pdfhttp://bioquimica.org.br/revista/ojs/index.php/REB/article/view/63810.16923/reb.v14i3.638Revista de Ensino de Bioquímica; v. 14, n. 3 (2016): REB (Jul - Dez); 34 - 35Revista de Enseñanza de Bioquímica; v. 14, n. 3 (2016): REB (Jul - Dez); 34 - 35Journal of Biochemistry Education; v. 14, n. 3 (2016): REB (Jul - Dez); 34 - 35Revista de Ensino de Bioquímica; v. 14, n. 3 (2016): REB (Jul - Dez); 34 - 352318-8790reponame:Revista de Ensino de Bioquímicainstname:Sociedade Brasileira de Bioquímica e Biologia Molecular (SBBq)instacron:SBBQMenghttp://bioquimica.org.br/revista/ojs/index.php/REB/article/view/638/566/*ref*/Gerdes HH, Kaether C. Green fluorescent protein: applications in cell biology. Federation of European Biochemical Societies. 1996, 389. p. 44-7./*ref*/Zimmer M. Green Fluorescent Protein (GFP): Applications, Structure, and Related Photophysical Behavior. American Chemical Society. 2002; 102. p. 759−781./*ref*/Ehrmann MA, Scheyhing CH, Vogel RF. (2001). In vitro stability and expression of green fluorescent protein under high pressure conditions. Letters in Applied Microbiology. 2001; 32; 230-234./*ref*/Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene.1990; 96(1); 23-8./*ref*/Sambrook J, Fritsch E.F, Maniatis T. (1989). Gel electrophoresis of DNA. In: Sambrook, J., Fritsch, E.F. and Maniatis, T. (Eds.) Molecular Cloning: a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, chapter 6./*ref*/Hochuli E, Dbeli H, Schacher A. (1987). New metal chelate adsorbent selective for proteins and peptide containing neighbouring histidine residues. J Chromatogry. 1987; 411: 177–184. [7] Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970; 227: 680-685. doi: 10.1038/227680a0./*ref*/Dieryck W, Noubhani AM, Coulon D, Santarelli X.. Cloning, expression and two-step purification of recombinant His-tag enhanced green fluorescent protein over-expressed in Escherichia coli. Journal of Chromatograpy. 2003; 726: 153-9./*ref*/Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992; 111(2): 229-33./*ref*/Zech K, Frei RW. Selective sample handling and detection in high performance liquid chromatography. Journal of Chromatograph library. 1989; 39./*ref*/Porath J, Jan C, Ingmar O, Greta B Metal chelate affinity chromatography, a new approach to protein fractionation. Nature. 1975; 258: 598-599./*ref*/Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein Measurement with the Folin Phenol Reagent. Journal of Biological Chemistry, 1951; 193: 265-275./*ref*/Wang L, Quan C, Liu B, Xu Y, Zhao P, Xiong W, Fan S. (2013). Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus. Int. J. Mol. Sci. 2013; 18470-18487; doi: 10.3390/ijms140918470./*ref*/Kaplow I M, Singh R, Friedman A, Bakal C, Perrimon N, Berger B. (2009). Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates. Nature methods. 2009; 6(7): 477-8.AustraliaDireitos autorais 2016 Revista de Ensino de Bioquímicahttp://creativecommons.org/licenses/by-nc-sa/4.0info:eu-repo/semantics/openAccess2022-03-25T17:24:46Zoai:ojs.bioquimica.org.br:article/638Revistahttp://bioquimica.org.br/revista/ojs/index.php/REBONGhttp://bioquimica.org.br/revista/ojs/index.php/REB/oaicontato@bioquimica.org.br||ensinodebioquimica@gmail.com2318-87901677-2318opendoar:2022-03-25T17:24:46Revista de Ensino de Bioquímica - Sociedade Brasileira de Bioquímica e Biologia Molecular (SBBq)false
dc.title.none.fl_str_mv Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia
Purificação de Proteína verde fluorescente por meio de Cromatografia de Afinidade com Íon Metal Imobilizado (IMAC) e sua relevância para estudantes de Ciências Biomédicas durante aulas de Bioquímica na Universidade La Trobe – Austrália
title Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia
spellingShingle Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia
de Melo Silva, Alex Jose José
practical class, purification
GFP; Purification; IMAC
Aulas práticas
GFP; Purificação; IMAC
title_short Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia
title_full Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia
title_fullStr Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia
title_full_unstemmed Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia
title_sort Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia
author de Melo Silva, Alex Jose José
author_facet de Melo Silva, Alex Jose José
Alves, Lumar Lucena
Pakay, Julian
author_role author
author2 Alves, Lumar Lucena
Pakay, Julian
author2_role author
author
dc.contributor.none.fl_str_mv

dc.contributor.author.fl_str_mv de Melo Silva, Alex Jose José
Alves, Lumar Lucena
Pakay, Julian
dc.subject.por.fl_str_mv practical class, purification
GFP; Purification; IMAC
Aulas práticas
GFP; Purificação; IMAC
topic practical class, purification
GFP; Purification; IMAC
Aulas práticas
GFP; Purificação; IMAC
description This work was performed as an integrated practical of a Biomedical Science undergraduate course of Biochemistry subject, in order to demonstrate used techniques to purify of Green Fluorescent Protein (GFP). To perform the experiments the main methodology applied was the by immobilized metal affinity chromatography (IMAC).  The open reading frame for enhanced GFP was sub-cloned into the pQE30 expression vector. The subsequent production of protein tagged N-terminally with hexahistidine, facilitated its purification by IMAC.  An approximate 3-fold purification of GFP was achieved. Thus, the students who completed the course gained significant experience related to fundamental techniques in molecular cloning and a sound basis in the principles of recombinant protein expression and purification.
publishDate 2016
dc.date.none.fl_str_mv 2016-12-15
dc.type.none.fl_str_mv




dc.type.driver.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
Practical
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://bioquimica.org.br/revista/ojs/index.php/REB/article/view/638
10.16923/reb.v14i3.638
url http://bioquimica.org.br/revista/ojs/index.php/REB/article/view/638
identifier_str_mv 10.16923/reb.v14i3.638
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv http://bioquimica.org.br/revista/ojs/index.php/REB/article/view/638/566
/*ref*/Gerdes HH, Kaether C. Green fluorescent protein: applications in cell biology. Federation of European Biochemical Societies. 1996, 389. p. 44-7.
/*ref*/Zimmer M. Green Fluorescent Protein (GFP): Applications, Structure, and Related Photophysical Behavior. American Chemical Society. 2002; 102. p. 759−781.
/*ref*/Ehrmann MA, Scheyhing CH, Vogel RF. (2001). In vitro stability and expression of green fluorescent protein under high pressure conditions. Letters in Applied Microbiology. 2001; 32; 230-234.
/*ref*/Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene.1990; 96(1); 23-8.
/*ref*/Sambrook J, Fritsch E.F, Maniatis T. (1989). Gel electrophoresis of DNA. In: Sambrook, J., Fritsch, E.F. and Maniatis, T. (Eds.) Molecular Cloning: a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, chapter 6.
/*ref*/Hochuli E, Dbeli H, Schacher A. (1987). New metal chelate adsorbent selective for proteins and peptide containing neighbouring histidine residues. J Chromatogry. 1987; 411: 177–184. [7] Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970; 227: 680-685. doi: 10.1038/227680a0.
/*ref*/Dieryck W, Noubhani AM, Coulon D, Santarelli X.. Cloning, expression and two-step purification of recombinant His-tag enhanced green fluorescent protein over-expressed in Escherichia coli. Journal of Chromatograpy. 2003; 726: 153-9.
/*ref*/Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992; 111(2): 229-33.
/*ref*/Zech K, Frei RW. Selective sample handling and detection in high performance liquid chromatography. Journal of Chromatograph library. 1989; 39.
/*ref*/Porath J, Jan C, Ingmar O, Greta B Metal chelate affinity chromatography, a new approach to protein fractionation. Nature. 1975; 258: 598-599.
/*ref*/Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein Measurement with the Folin Phenol Reagent. Journal of Biological Chemistry, 1951; 193: 265-275.
/*ref*/Wang L, Quan C, Liu B, Xu Y, Zhao P, Xiong W, Fan S. (2013). Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus. Int. J. Mol. Sci. 2013; 18470-18487; doi: 10.3390/ijms140918470.
/*ref*/Kaplow I M, Singh R, Friedman A, Bakal C, Perrimon N, Berger B. (2009). Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates. Nature methods. 2009; 6(7): 477-8.
dc.rights.driver.fl_str_mv Direitos autorais 2016 Revista de Ensino de Bioquímica
http://creativecommons.org/licenses/by-nc-sa/4.0
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Direitos autorais 2016 Revista de Ensino de Bioquímica
http://creativecommons.org/licenses/by-nc-sa/4.0
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.coverage.none.fl_str_mv Australia
dc.publisher.none.fl_str_mv Sociedade Brasileira de Bioquímica e Biologia Molecular - SBBq
publisher.none.fl_str_mv Sociedade Brasileira de Bioquímica e Biologia Molecular - SBBq
dc.source.none.fl_str_mv Revista de Ensino de Bioquímica; v. 14, n. 3 (2016): REB (Jul - Dez); 34 - 35
Revista de Enseñanza de Bioquímica; v. 14, n. 3 (2016): REB (Jul - Dez); 34 - 35
Journal of Biochemistry Education; v. 14, n. 3 (2016): REB (Jul - Dez); 34 - 35
Revista de Ensino de Bioquímica; v. 14, n. 3 (2016): REB (Jul - Dez); 34 - 35
2318-8790
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repository.name.fl_str_mv Revista de Ensino de Bioquímica - Sociedade Brasileira de Bioquímica e Biologia Molecular (SBBq)
repository.mail.fl_str_mv contato@bioquimica.org.br||ensinodebioquimica@gmail.com
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