Abstract
The expansion of the genetic code has become a valuable tool for molecular biology, biochemistry, and biotechnology. The pyrrolysyl-tRNA synthetase (PylRS) variants with their cognate tRNAPyl derived from methanogenic archaea of the genus Methanosarcina are the most popular tools for ribosomally mediated site-specific and proteome-wide statistical incorporation of noncanonical amino acids (ncAAs) into proteins. The incorporation of ncAAs can be used for numerous biotechnological and even therapeutically relevant applications. Here we present a protocol of engineering PylRS for novel substrates with unique chemical functionalities. These functional groups can act as intrinsic probes, especially in complex biological environments such as mammalian cells, tissues, and even whole animals.
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