دورية
Generating Efficient Methanomethylophilus alvusPyrrolysyl-tRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids
العنوان: | Generating Efficient Methanomethylophilus alvusPyrrolysyl-tRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids |
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المؤلفون: | Avila-Crump, Savanna, Hemshorn, Marcus L., Jones, Chloe M., Mbengi, Lea, Meyer, Kyle, Griffis, Joshua A., Jana, Subhashis, Petrina, Grace E., Pagar, Vinayak V., Karplus, P. Andrew, Petersson, E. James, Perona, John J., Mehl, Ryan A., Cooley, Richard B. |
المصدر: | ACS Chemical Biology; 20220101, Issue: Preprints |
مستخلص: | Genetic code expansion (GCE) technologies commonly use the pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylpairs from Methanosarcina mazei(Mm) and Methanosarcina barkeri(Mb) for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Recently a homologous PylRS/tRNAPylpair from CandidatusMethanomethylophilus alvusMx1201(Ma) was developed that, lacking the N-terminal tRNA-recognition domain of most PylRSs, overcomes insolubility, instability, and proteolysis issues seen with Mb/MmPylRSs. An open question is how to alter MaPylRS specificity to encode specific ncAAs with high efficiency. Prior work focused on “transplanting” ncAA substrate specificity by reconstructing the same active site mutations found in functional Mm/MbPylRSs in MaPylRS. Here, we found that this strategy produced low-efficiency MaPylRSs for encoding three structurally diverse ncAAs: acridonyl-alanine (Acd), 3-nitro-tyrosine, and m-methyl-tetrazinyl-phenylalanine (Tet3.0-Me). On the other hand, efficient MaPylRS variants were generated by a conventional life/death selection process from a large library of active site mutants: for Acd encoding, one variant was highly functional in HEK293T cells at just 10 μM Acd; for nitroY encoding, two variants also encoded 3-chloro, 3-bromo-, and 3-iodo-tyrosine at high efficiency; and for Tet-3.0-Me, all variants were more functional at lower ncAA concentrations. All MaPylRS variants identified through selection had at least two different active site residues when compared with their MbPylRS counterparts. We conclude that Maand Mm/MbPylRSs are sufficiently different that “active site transplantation” yields suboptimal MaGCE systems. This work establishes a paradigm for expanding the utility of the promising MaPylRS/tRNAPylGCE platform. |
قاعدة البيانات: | Supplemental Index |
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