Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells.

Published: 19 Jul 2018 | Version 1 | DOI: 10.17632/bnm4x5vjrs.1
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Description of this data

Genetic code expansion via stop codon suppression is a versatile tool for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

Experiment data files

Latest version

  • Version 1

    2018-07-19

    Published: 2018-07-19

    DOI: 10.17632/bnm4x5vjrs.1

    Cite this dataset

    Elsässer, Simon; Meineke, Birthe (2018), “Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells.”, Mendeley Data, v1 http://dx.doi.org/10.17632/bnm4x5vjrs.1

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Institutions

Karolinska Institutet Department of Medical Biochemistry and Biophysics

Categories

Plasmid, Synthetic Biology, Protein Engineering, Mammalian Cell, Genetic Code

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