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

Wellington P Oliveira-Souza, Fellipe Bronze, Jaap Broos, Marcelo F. M. Marcondes*, Vitor Oliveira

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

2 Citations (Scopus)
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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.

Original languageEnglish
Article numberj.bbrc.2017.08.111
Pages (from-to)343-348
Number of pages6
JournalBiochemical and Biophysical Research Communications
Issue number3
Early online date30-Aug-2017
Publication statusPublished - 21-Oct-2017


  • Unnatural amino acid
  • Intrinsic fluorescence
  • Tryptophan analogs
  • Protein labeling
  • HOST

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