Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags
Introducing non-canonical amino acids (ncAAs) by engineered orthogonal pairs of aminoacyl-tRNA synthetases and tRNAs has proven to be a highly useful tool for the expansion of the genetic code. Pyrrolysyl-tRNA synthetase (PylRS) from methanogenic archaeal and bacterial species is particularly attrac...
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Frontiers Media S.A.
2021-12-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2021.807438/full |
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| author | Nikolaj G. Koch Nikolaj G. Koch Tobias Baumann Nediljko Budisa Nediljko Budisa |
| author_facet | Nikolaj G. Koch Nikolaj G. Koch Tobias Baumann Nediljko Budisa Nediljko Budisa |
| author_sort | Nikolaj G. Koch |
| collection | DOAJ |
| description | Introducing non-canonical amino acids (ncAAs) by engineered orthogonal pairs of aminoacyl-tRNA synthetases and tRNAs has proven to be a highly useful tool for the expansion of the genetic code. Pyrrolysyl-tRNA synthetase (PylRS) from methanogenic archaeal and bacterial species is particularly attractive due to its natural orthogonal reactivity in bacterial and eukaryotic cells. However, the scope of such a reprogrammed translation is often limited, due to low yields of chemically modified target protein. This can be the result of substrate specificity engineering, which decreases the aminoacyl-tRNA synthetase stability and reduces the abundance of active enzyme. We show that the solubility and folding of these engineered enzymes can become a bottleneck for the production of ncAA-containing proteins in vivo. Solubility tags derived from various species provide a strategy to remedy this issue. We find the N-terminal fusion of the small metal binding protein from Nitrosomonas europaea to the PylRS sequence to improve enzyme solubility and to boost orthogonal translation efficiency. Our strategy enhances the production of site-specifically labelled proteins with a variety of engineered PylRS variants by 200–540%, and further allows triple labeling. Even the wild-type enzyme gains up to 245% efficiency for established ncAA substrates. |
| first_indexed | 2024-12-13T13:18:03Z |
| format | Article |
| id | doaj.art-1ff7690580b849d6823dc527d1fdfef1 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-12-13T13:18:03Z |
| publishDate | 2021-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-1ff7690580b849d6823dc527d1fdfef12022-12-21T23:44:29ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852021-12-01910.3389/fbioe.2021.807438807438Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility TagsNikolaj G. Koch0Nikolaj G. Koch1Tobias Baumann2Nediljko Budisa3Nediljko Budisa4Biokatalyse, Institut für Chemie, Technische Universität Berlin, Berlin, GermanyBioanalytik, Institut für Biotechnologie, Technische Universität Berlin, Berlin, GermanyBiokatalyse, Institut für Chemie, Technische Universität Berlin, Berlin, GermanyBiokatalyse, Institut für Chemie, Technische Universität Berlin, Berlin, GermanyChemical Synthetic Biology, Department of Chemistry, University of Manitoba, Winnipeg, MB, CanadaIntroducing non-canonical amino acids (ncAAs) by engineered orthogonal pairs of aminoacyl-tRNA synthetases and tRNAs has proven to be a highly useful tool for the expansion of the genetic code. Pyrrolysyl-tRNA synthetase (PylRS) from methanogenic archaeal and bacterial species is particularly attractive due to its natural orthogonal reactivity in bacterial and eukaryotic cells. However, the scope of such a reprogrammed translation is often limited, due to low yields of chemically modified target protein. This can be the result of substrate specificity engineering, which decreases the aminoacyl-tRNA synthetase stability and reduces the abundance of active enzyme. We show that the solubility and folding of these engineered enzymes can become a bottleneck for the production of ncAA-containing proteins in vivo. Solubility tags derived from various species provide a strategy to remedy this issue. We find the N-terminal fusion of the small metal binding protein from Nitrosomonas europaea to the PylRS sequence to improve enzyme solubility and to boost orthogonal translation efficiency. Our strategy enhances the production of site-specifically labelled proteins with a variety of engineered PylRS variants by 200–540%, and further allows triple labeling. Even the wild-type enzyme gains up to 245% efficiency for established ncAA substrates.https://www.frontiersin.org/articles/10.3389/fbioe.2021.807438/fullgenetic code expansionpyrrolysyl-tRNA synthetasenon-canonical amino acidprotein engineeringsolubility tagsstop codon suppression |
| spellingShingle | Nikolaj G. Koch Nikolaj G. Koch Tobias Baumann Nediljko Budisa Nediljko Budisa Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags Frontiers in Bioengineering and Biotechnology genetic code expansion pyrrolysyl-tRNA synthetase non-canonical amino acid protein engineering solubility tags stop codon suppression |
| title | Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags |
| title_full | Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags |
| title_fullStr | Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags |
| title_full_unstemmed | Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags |
| title_short | Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags |
| title_sort | efficient unnatural protein production by pyrrolysyl trna synthetase with genetically fused solubility tags |
| topic | genetic code expansion pyrrolysyl-tRNA synthetase non-canonical amino acid protein engineering solubility tags stop codon suppression |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2021.807438/full |
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