Biophysical Society Thematic Meeting - October 25-30, 2015

Polymers and Self Assembly: From Biology to Nanomaterials Poster Session I

8-POS Board 8 Efficient Bio Incorporation of Bulky-Tryptophan Analogs in Recombinant Proteins Expressed in E. coli: Obtaining α-Synuclein Labeled with 5-Hydroxy-Tryptophan Fellipe Bronze 1 , Wellington P. Souza 1 , Marcelo Marcondes 1,2 , Jaap Broos 2 , Vitor Oliveira 1 . 1 UNIFESP, Sao Paulo, Brazil, 2 University of Groningen, Groningen, Netherlands. Α-Synuclein oligomerization/aggregation has been implicated in many progressive neurodegenerative diseases. Labeling the α-synuclein with spectroscopic probes has been a useful tool to investigate the oligomerization/aggregation of this protein in vitro and even in vivo. Among the available labeling methods, biosynthetic incorporation of non-canonic amino acids is an attractive strategy to introduce new properties in recombinant proteins. In the present work we describe an efficient method to bio incorporate bulky tryptophan (Trp) analogs in proteins expressed in E. coli. Furthermore, this method is compatible with bacterial vectors based on the T7-RNA polymerase/promoter that are widely used for protein expression. The first target protein for this study was the recombinant α-synuclein mutant (V26W) cloned in one T7-based vector, the pET26b. The probe to be incorporated in this target was 5-hydroxy- Tryptophan (5HW) in the place of the regular W at position 26 introduced by mutagenesis (mutation V26W). In fact, there are few descriptions that 5-hydroxy-tryptophan (5HW) could be bio incorporated in recombinant proteins in E. coli, and with a large range of efficiencies. Nevertheless, we verified that such inefficient 5HW bio incorporation in T7-based systems is because that the central T7-RNA polymerase in this system has the same control as the target protein, resulting in formation of inactive T7-RNA polymerase. To overcome this problem we developed a two-step expression protocol that resulted in incorporation efficiencies of 5HW higher than 90% in α-synuclein mutant (V26W). The results also show other possibilities for the bio incorporation of bulkier tryptophan analogs (Ex: nitro-Trp, 5Br-Trp, F-Trp). Bulkier Trp analogs may not be recognized by regular E. coli Tryptophanyl-tRNA synthetase. But, the co-expression of a more permissive synthetase makes possible the incorporation of such bulkier Trp analogs.

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