A Facile Method for Producing Selenocysteine‐Containing Proteins |
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| Authors: | Dr. Takahito Mukai Dr. Anastasia Sevostyanova Dr. Tateki Suzuki Dr. Xian Fu Prof. Dieter Söll |
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| Affiliation: | 1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA;2. Department of Chemistry, Yale University, New Haven, CT, USA |
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| Abstract: | Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNASer. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDHH with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNAUTu system offers a new selenoprotein engineering platform. |
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| Keywords: | genetic code expansion protein engineering selenocysteine selenoproteins synthetic biology |
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