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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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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Author(s):
Oliveira-Souza, Wellington P. [1] ; Bronze, Fellipe [1] ; Broos, Jaap [2] ; Marcondes, Marcelo F. M. [1, 2] ; Oliveira, Vitor [1]
Total Authors: 5
Affiliation:
[1] Univ Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Pedro de Toledo 669, 7 Andar, Sao Paulo - Brazil
[2] Univ Groningen, Groningen Biomol Sci & Biotechnol Inst, Biophys Chem, Nijenborgh 7, NL-9747 AG Groningen - Netherlands
Total Affiliations: 2
Document type: Journal article
Source: Biochemical and Biophysical Research Communications; v. 492, n. 3, p. 343-348, OCT 21 2017.
Web of Science Citations: 1
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. (AU)

FAPESP's process: 14/20847-0 - Site-specific incorporation of non-natural amino acids in proteins: development of new incorporation technologies and the use in the study of proteins
Grantee:Vitor Marcelo Silveira Bueno Brandão de Oliveira
Support Opportunities: Regular Research Grants
FAPESP's process: 11/20941-9 - DEVELOPMENT AND USE OF FLUORESCENCE TECHNIQUES FOR THE STUDY OF PROTEIN CONFORMATIONAL CHANGES
Grantee:Marcelo Ferreira Marcondes Machado
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 14/00661-0 - Development and use of fluorescence techniques for the study of protein conformational changes
Grantee:Marcelo Ferreira Marcondes Machado
Support Opportunities: Scholarships abroad - Research Internship - Post-doctor