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

Autor(es)

Oliveira-Souza, Wellington P. ^[1] ; Bronze, Fellipe ^[1] ; Broos, Jaap ^[2] ; Marcondes, Marcelo F. M. ^{[1, 2]} ; Oliveira, Vitor ^[1]

Afiliação do(s) autor(es)

^[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

• Tipo de documento: Artigo Científico
• Fonte: Biochemical and Biophysical Research Communications; v. 492, n. 3, p. 343-348, OCT 21 2017.
• Citações Web of Science: 1

Resumo

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)

• Processo FAPESP: 14/20847-0 - Incorporação sítio específica de aminoácidos não naturais em proteínas: desenvolvimento de novas tecnologias de incorporação e aplicação no estudo de proteínas
• Beneficiário: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 11/20941-9 - Desenvolvimento e utilização de técnicas de fluorescência para o estudo de alterações conformacionais em proteínas
• Beneficiário: Marcelo Ferreira Marcondes Machado
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 14/00661-0 - Desenvolvimento e utilização de técnicas de fluorescência para o estudo de alterações conformacionais em proteínas
• Beneficiário: Marcelo Ferreira Marcondes Machado
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Pós-Doutorado