Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery
3,4-Dihydroxy-L-phenylalanine (DOPA) is a promising noncanonical amino acid (ncAA) that introduces novel catechol chemical features into proteins, expanding their functional potential. However, the most common approach to incorporating ncAAs into proteins relies on stop codon suppression, which is o...
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MDPI AG
2025-03-01
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| author | Youhui Yang Yingchen Wang Zhaoguan Wang Hao Qi |
| author_facet | Youhui Yang Yingchen Wang Zhaoguan Wang Hao Qi |
| author_sort | Youhui Yang |
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| description | 3,4-Dihydroxy-L-phenylalanine (DOPA) is a promising noncanonical amino acid (ncAA) that introduces novel catechol chemical features into proteins, expanding their functional potential. However, the most common approach to incorporating ncAAs into proteins relies on stop codon suppression, which is often limited by the competition of endogenous translational termination machinery. Here, we employed a special in vitro protein expression system that facilitates the efficiency of DOPA incorporation into proteins by removing essential Class I peptide release factors through targeted degradation. In the absence of both RF1 and RF2, we successfully demonstrated DOPA incorporation at all three stop codons (TAG, TAA, and TGA). By optimizing the concentration of engineered DOPA-specific aminoacyl-tRNA synthetase (DOPARS), DOPA, and DNA template, we achieved a synthesis yield of 2.24 µg of sfGFP with 100% DOPA incorporation in a 20 μL reaction system. DOPARS exhibited a dissociation constant (<i>Kd</i>) of 11.7 μM for DOPA but showed no detectable binding to its native counterpart, tyrosine. Additionally, DOPA was successfully incorporated into a reverse transcriptase, which interfered with its activity. This system demonstrates a fast and efficient approach for precise DOPA incorporation into proteins, paving the way for advanced protein engineering applications. |
| format | Article |
| id | doaj-art-d015d2ca6f2d41d0b2c88962d496acaa |
| institution | OA Journals |
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| language | English |
| publishDate | 2025-03-01 |
| publisher | MDPI AG |
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| series | Biomolecules |
| spelling | doaj-art-d015d2ca6f2d41d0b2c88962d496acaa2025-08-20T02:11:22ZengMDPI AGBiomolecules2218-273X2025-03-0115338210.3390/biom15030382Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination MachineryYouhui Yang0Yingchen Wang1Zhaoguan Wang2Hao Qi3School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, ChinaSchool of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, ChinaSchool of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, ChinaSchool of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China3,4-Dihydroxy-L-phenylalanine (DOPA) is a promising noncanonical amino acid (ncAA) that introduces novel catechol chemical features into proteins, expanding their functional potential. However, the most common approach to incorporating ncAAs into proteins relies on stop codon suppression, which is often limited by the competition of endogenous translational termination machinery. Here, we employed a special in vitro protein expression system that facilitates the efficiency of DOPA incorporation into proteins by removing essential Class I peptide release factors through targeted degradation. In the absence of both RF1 and RF2, we successfully demonstrated DOPA incorporation at all three stop codons (TAG, TAA, and TGA). By optimizing the concentration of engineered DOPA-specific aminoacyl-tRNA synthetase (DOPARS), DOPA, and DNA template, we achieved a synthesis yield of 2.24 µg of sfGFP with 100% DOPA incorporation in a 20 μL reaction system. DOPARS exhibited a dissociation constant (<i>Kd</i>) of 11.7 μM for DOPA but showed no detectable binding to its native counterpart, tyrosine. Additionally, DOPA was successfully incorporated into a reverse transcriptase, which interfered with its activity. This system demonstrates a fast and efficient approach for precise DOPA incorporation into proteins, paving the way for advanced protein engineering applications.https://www.mdpi.com/2218-273X/15/3/382noncanonical amino acids3,4-dihydroxy-L-phenylalaninesfGFPaminoacyl-tRNA synthetase |
| spellingShingle | Youhui Yang Yingchen Wang Zhaoguan Wang Hao Qi Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery Biomolecules noncanonical amino acids 3,4-dihydroxy-L-phenylalanine sfGFP aminoacyl-tRNA synthetase |
| title | Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery |
| title_full | Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery |
| title_fullStr | Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery |
| title_full_unstemmed | Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery |
| title_short | Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery |
| title_sort | efficient incorporation of dopa into proteins free from competition with endogenous translation termination machinery |
| topic | noncanonical amino acids 3,4-dihydroxy-L-phenylalanine sfGFP aminoacyl-tRNA synthetase |
| url | https://www.mdpi.com/2218-273X/15/3/382 |
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