Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisation
| Autor(a) principal: | |
|---|---|
| Data de Publicação: | 2013 |
| 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/1822/31033 |
Resumo: | The authors wish to thank Prof. Dorit Schuller and Inês Mendes for the use of and assistance with the HPLC column and all the technical staff at the CBMA for their skilful technical assistance. |
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Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisationSELPbatch productionBiopolymersSilk-elastin like polymerspET-E. coli BL21(DE3)Ciências Naturais::Ciências BiológicasScience & TechnologyThe authors wish to thank Prof. Dorit Schuller and Inês Mendes for the use of and assistance with the HPLC column and all the technical staff at the CBMA for their skilful technical assistance.Background: Silk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system. Results: This study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6–7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained. Conclusions: We have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21 (DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production. Keywords: Biopolymers, Silk-elastin like polymers, pET-E. coli BL21(DE3), Batch productionThis work was financed by the European Commission, via the 7th Framework Programme Project EcoPlast (FP7-NMP-2009-SME-3, collaborative project number 246176), by FEDER through POFC – COMPETE and by national funds from Fundação para a Ciência e Tecnologia (FCT) through PEst project C/BIA/UI4050/2011C/BIA/UI4050/2011. AC is a recipient of FCT grant SFRH/ BD/75882/2011. TC is thankful to FCT for its support through Programa Ciência 2008.Fundação para a Ciência e a Tecnologia (FCT)BioMed Central (BMC)Universidade do MinhoCollins, TonySilva, João AzevedoCosta, André daBranca, FernandoMachado, RaulCasal, Margarida2013-02-272013-02-27T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/31033engCollins et al.: Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation. Microbial Cell Factories 2013 12:21.1475-285910.1186/1475-2859-12-2123446293http://www.microbialcellfactories.com/content/12/1/21info: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:RCAAP2023-07-21T12:45:34Zoai:repositorium.sdum.uminho.pt:1822/31033Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:43:25.800386Repositório 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 |
Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisation |
| title |
Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisation |
| spellingShingle |
Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisation Collins, Tony SELP batch production Biopolymers Silk-elastin like polymers pET-E. coli BL21(DE3) Ciências Naturais::Ciências Biológicas Science & Technology |
| title_short |
Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisation |
| title_full |
Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisation |
| title_fullStr |
Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisation |
| title_full_unstemmed |
Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisation |
| title_sort |
Batch production of a silk-elastin-like protein in E. coli BL21(DE3) : key parameters for optimisation |
| author |
Collins, Tony |
| author_facet |
Collins, Tony Silva, João Azevedo Costa, André da Branca, Fernando Machado, Raul Casal, Margarida |
| author_role |
author |
| author2 |
Silva, João Azevedo Costa, André da Branca, Fernando Machado, Raul Casal, Margarida |
| author2_role |
author author author author author |
| dc.contributor.none.fl_str_mv |
Universidade do Minho |
| dc.contributor.author.fl_str_mv |
Collins, Tony Silva, João Azevedo Costa, André da Branca, Fernando Machado, Raul Casal, Margarida |
| dc.subject.por.fl_str_mv |
SELP batch production Biopolymers Silk-elastin like polymers pET-E. coli BL21(DE3) Ciências Naturais::Ciências Biológicas Science & Technology |
| topic |
SELP batch production Biopolymers Silk-elastin like polymers pET-E. coli BL21(DE3) Ciências Naturais::Ciências Biológicas Science & Technology |
| description |
The authors wish to thank Prof. Dorit Schuller and Inês Mendes for the use of and assistance with the HPLC column and all the technical staff at the CBMA for their skilful technical assistance. |
| publishDate |
2013 |
| dc.date.none.fl_str_mv |
2013-02-27 2013-02-27T00:00:00Z |
| 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 |
http://hdl.handle.net/1822/31033 |
| url |
http://hdl.handle.net/1822/31033 |
| dc.language.iso.fl_str_mv |
eng |
| language |
eng |
| dc.relation.none.fl_str_mv |
Collins et al.: Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation. Microbial Cell Factories 2013 12:21. 1475-2859 10.1186/1475-2859-12-21 23446293 http://www.microbialcellfactories.com/content/12/1/21 |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
| dc.publisher.none.fl_str_mv |
BioMed Central (BMC) |
| publisher.none.fl_str_mv |
BioMed Central (BMC) |
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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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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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