On the efficient bio-incorporation of 5-hydroxy-tryptophan in recombinant proteins expressed in Escherichia coli with T7 RNA polymerase-based vectors

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dc.citation.issue3
dc.citation.volume492
dc.contributor.authorOliveira-Souza, Wellington P. [UNIFESP]
dc.contributor.authorBronze, Fellipe [UNIFESP]
dc.contributor.authorBroos, Jaap
dc.contributor.authorMarcondes, Marcelo F. M. [UNIFESP]
dc.contributor.authorOliveira, Vitor [UNIFESP]
dc.coverageSan Diego
dc.date.accessioned2020-08-04T13:39:49Z
dc.date.available2020-08-04T13:39:49Z
dc.date.issued2017
dc.description.abstractBiosynthetic incorporation of non-canonic amino acids is an attractive strategy to introduce new properties in recombinant proteins. Trp analogs can be incorporated in recombinant proteins replacing regular Trp during protein translation into a Trp-auxotrophic cell host. This straightforward method however, is limited to few analogs recognized and accepted by the cellular protein production machinery. 5-hydroxy-tryptophan (50H-Trp) can be bio-incorporated using E. coli as expression host howeveren
dc.description.abstractwe have experienced very low incorporation yields - amount of protein containing regular Trp/amount of protein containing the Trp analog during expressions of 50H-Trp labeled proteins. Furthermore, this low incorporation yield were verified especially when the widely-used vectors based on the 17 RNA polymerase were used. Testing different 50H-Trp incorporation protocols we verified that in these T7 based systems, the production of the T7 RNA polymerase is driven by the same elements lac promoter/IPTG as the target protein. Consequently, the bio-incorporation of the 50H-Trp residues also occurs in this crucial enzyme, but, the produced T7 RNA polymerase labeled with 50H-Trp is inactive or much less active. In the present work, we describe an efficient method to overcome this mentioned problem and bio-incorporate 50H-Trp in proteins expressed in E. coli., using vectors based on the 17 RNA polymerase-T7 promoter. The two-step induction protocol here described showed incorporation efficiencies of 50H-Trp higher than 90%. (C) 2017 Elsevier Inc. All rights reserved.en
dc.description.affiliationUniv Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Pedro de Toledo 669,7 Andar, Sao Paulo, Brazil
dc.description.affiliationUniv Groningen, Groningen Biomol Sci & Biotechnol Inst, Biophys Chem, Nijenborgh 7, NL-9747 AG Groningen, Netherlands
dc.description.affiliationUnifespUniv Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Pedro de Toledo 669,7 Andar, Sao Paulo, Brazil
dc.description.sourceWeb of Science
dc.description.sponsorshipFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)
dc.description.sponsorshipConselho nacional de pesquisa, ciencia e tecnologia (CNPq)
dc.description.sponsorshipIDFAPESP: 2014/20847-0
dc.description.sponsorshipIDFAPESP: 2011/20941-9
dc.description.sponsorshipIDFAPESP: 2014/00661-0
dc.description.sponsorshipIDCNPq: 458010/2014-6
dc.description.sponsorshipIDCNPq: 308111/2014-1
dc.format.extent343-348
dc.identifierhttp://dx.doi.org/10.1016/j.bbrc.2017.08.111
dc.identifier.citationBiochemical And Biophysical Research Communications. San Diego, v. 492, n. 3, p. 343-348, 2017.
dc.identifier.doi10.1016/j.bbrc.2017.08.111
dc.identifier.issn0006-291X
dc.identifier.urihttps://repositorio.unifesp.br/handle/11600/57134
dc.identifier.wosWOS:000411424300010
dc.language.isoeng
dc.publisherAcademic Press Inc Elsevier Science
dc.relation.ispartofBiochemical And Biophysical Research Communications
dc.rightsinfo:eu-repo/semantics/restrictedAccess
dc.subjectUnnatural amino aciden
dc.subjectIntrinsic fluorescenceen
dc.subjectTryptophan analogsen
dc.subjectProtein labelingen
dc.titleOn the efficient bio-incorporation of 5-hydroxy-tryptophan in recombinant proteins expressed in Escherichia coli with T7 RNA polymerase-based vectorsen
dc.typeinfo:eu-repo/semantics/article
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