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

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 howeverwe 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.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho nacional de pesquisa, ciencia e tecnologia (CNPq)Univ Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Pedro de Toledo 669,7 Andar, Sao Paulo, BrazilUniv Groningen, Groningen Biomol Sci & Biotechnol Inst, Biophys Chem, Nijenborgh 7, NL-9747 AG Groningen, NetherlandsUniv Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Pedro de Toledo 669,7 Andar, Sao Paulo, BrazilFAPESP: 2014/20847-0FAPESP: 2011/20941-9FAPESP: 2014/00661-0CNPq: 458010/2014-6CNPq: 308111/2014-1Web of Scienc