TY - JOUR
T1 - A Facile Method for Producing Selenocysteine-Containing Proteins
AU - Mukai, Takahito
AU - Sevostyanova, Anastasia
AU - Suzuki, Tateki
AU - Fu, Xian
AU - Söll, Dieter
N1 - Funding Information:
We thank Anna Merkuryev for help with SelA engineering and Navin Rauniyar, Ana Crnković, and Omer Ad for dedicated efforts on the MS analyses. T.M. is a Japan Society for the Promotion of Science postdoctoral fellow for research abroad. We acknowledge support of this work from the US National Institutes of Health grants (R01GM22854 and R35GM122560 to D.S.); the Department of Energy DE-FG02-98ER20311 grant (to D.S.) supported the genetic experiments.
Publisher Copyright:
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
PY - 2018/6/11
Y1 - 2018/6/11
N2 - 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.
AB - 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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U2 - 10.1002/anie.201713215
DO - 10.1002/anie.201713215
M3 - Article
C2 - 29631320
AN - SCOPUS:85046622061
SN - 1433-7851
VL - 57
SP - 7215
EP - 7219
JO - Angewandte Chemie - International Edition
JF - Angewandte Chemie - International Edition
IS - 24
ER -