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

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

Author Oliveira-Souza, Wellington P. Autor UNIFESP Google Scholar
Bronze, Fellipe Autor UNIFESP Google Scholar
Broos, Jaap Google Scholar
Marcondes, Marcelo F. M. Autor UNIFESP Google Scholar
Oliveira, Vitor Autor UNIFESP Google Scholar
Abstract Biosynthetic 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 however

we 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.
Keywords Unnatural amino acid
Intrinsic fluorescence
Tryptophan analogs
Protein labeling
xmlui.dri2xhtml.METS-1.0.item-coverage San Diego
Language English
Sponsor Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)
Conselho nacional de pesquisa, ciencia e tecnologia (CNPq)
Grant number FAPESP: 2014/20847-0
FAPESP: 2011/20941-9
FAPESP: 2014/00661-0
CNPq: 458010/2014-6
CNPq: 308111/2014-1
Date 2017
Published in Biochemical And Biophysical Research Communications. San Diego, v. 492, n. 3, p. 343-348, 2017.
ISSN 0006-291X (Sherpa/Romeo, impact factor)
Publisher Academic Press Inc Elsevier Science
Extent 343-348
Origin http://dx.doi.org/10.1016/j.bbrc.2017.08.111
Access rights Closed access
Type Article
Web of Science ID WOS:000411424300010
URI https://repositorio.unifesp.br/handle/11600/57134

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