Expanding the Library and Substrate Diversity of the Pyrrolysyl-tRNA Synthetase to Incorporate Unnatural Amino Acids Containing Conjugated Rings

摘要

Genetically encoded unnatural amino acids (UAAs), incorporated using orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs, have greatly facilitated the study and manipulation of proteins and biological processes.~([1-3]) UAAs are available with a diversity far exceeding that of nature's canonical amino acids. Through site-directed incorporation of UAAs with specific physical or chemical characteristics, proteins can be engineered with novel properties.~([3]) A large number of distinct UAAs have been incorporated with tRNA/aaRS pairs evolved from the original tyrosyl amber suppressor tRNA (tRNA_(CUA)~(Tyr)) and its cognate tyrosyl-tRNA synthetase (TyrRS).~([4]) In recent years, the pyrrolysyl amber suppressor tRNA and cognate synthetase (tRNA_(CUA)~(Pyl)/PylRS) pair derived from the archaebacteria Methanosarcina mazei and M. barkeri has provided a convenient alternative. This pair is orthogonal to endogenous tRNA/aaRS pairs in both E. coli and mammalian cells,~([5-7]) and so mutant synthetases specific for UAAs can be evolved in a bacterial host and subsequently imported into to eukaryotic cells for UAA incorporation. A number of UAAs with structures similar to the native substrate pyrrolysine (Pyl) have been incorporated using the wild-type or engineered PylRS.~([3, 6, 8]) We have previously demonstrated that through replacement of a residue serving as the "gate keeper" for Pyl binding, the PylRS can be further engineered to incorporate UAAs with structures divergent from Pyl.~([9]) In these UAAs, the long aliphatic side chain and the core Ne-carbonyl group of Pyl is replaced by an aromatic ring, thereby allowing the incorporation of tyrosine or phenylalanine derivatives.~([9, 10])