A second, orthogonal pyrrolysyl-tRNA-synthetase pair for mammalian cells

pylrs and plt.png

Cloverleaf sructure of Mma and Mx1201 PylTs with grey cartoons of related PylRS showing predicted sites of recognition between enzyme and tRNA.

For the scientists

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.

Find the paper: https://pubs.acs.org/doi/10.1021/acschembio.8b00571

Supplementary Data: http://dx.doi.org/10.17632/bnm4x5vjrs.1

Addgene Plasmids: https://www.addgene.org/browse/article/28196809/

For the young scientists

  contributed by summer student Ruth Lappalainen

Apart from the 21 common amino acids used in virtually all proteins, chemists have come up with a much wider range of amino acids, not naturally present in cells, known as non-canonical amino acids (ncAA). These can be incorporated into proteins by tinkering with protein synthesis on the ribosome. During  translation, the ribosome decodes the genetic code using transfer RNAs (tRNAs).  The amino acid  to be incorporated by the ribosome is attached to a tRNA molecule with the help of enzymes known as aminoacyl-tRNA synthetases. These enzymes are extermely specific to their tRNA partner and as to which amino acid they will attach to it.

We developed a new method incorporating ncAAs into proteins of mammalian cells. This new method involves a previously uncharacterised tRNA/aminoacyl-tRNA synthetase pair from human gut archaeon Methanomethylophilus alvus, called Mx1201 PylRS/PyIT.  Mx1201 PylRS/PyIT normally incorporates a natural ncAA called pyrrolysyl and is also active and orthogonal in mammalian cells. Orthogonality here means that the pair does not react with the cell’s native tRNAs and aminoacyl-tRNA synthetases.  We have then engineered the new PylRS enzyme so that its ncAA range can be expanded.

Previously only the tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina (Mma PylRS/PylT) had been studied for pyrrolysyl incorporation. Since the two organisms have independently evolved in their respective habitats for most likely billions of years, Mx1201 PylRS/PyIT and Mma PylRS/PyIT they are actually quite different on the molecular level. This can be exploited in a way that each enzyme can be assigned a unique function in the cell so that they  can act independently (they are then ‘orthogonal’ to each other). We achieved orthogonality by mutation on Mx1201 PylT, forming two non cross-reactive PylRS/PylT pairs. These two pairs can now be used by the ribosome to incorporation two different ncAAs  at specific stop codons, in the same mammalian cell.


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