Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmid Construction
2.2. Genome Editing
2.3. Extract Preparation
2.4. Western Blot
2.5. Expression and Purification of the Proteins
2.6. In Vitro ncAA Incorporation
2.7. sfGFP Quantitative Expression
2.8. Mass Spectrometry Analysis
2.9. Isothermal Titration Calorimetry (ITC)
2.10. DOPA-Containing Protein Reverse Transcription Assay
3. Results
3.1. Establishment of CFUPS Without Class I Release Factor
3.2. Optimized Incorporation of DOPA in CFUPS
3.3. DOPA-Containing Protein Expression
3.4. Measurement of the Affinity of DOPARS for DOPA
3.5. Incorporation of L-DOPA into FrM4 RT in CFUPS
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
CFUPS | Cell-free unnatural protein synthesis |
ncAAs | Noncanonical amino acids |
GCE | Genetic code extension |
ITC | Isothermal titration calorimetry |
DOPARS | Methanocaldococcus jannaschii Tyrosyl-tRNA Synthetase |
DOPA | 3,4-Dihydroxy-L-phenylalanine |
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Yang, Y.; Wang, Y.; Wang, Z.; Qi, H. Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery. Biomolecules 2025, 15, 382. https://doi.org/10.3390/biom15030382
Yang Y, Wang Y, Wang Z, Qi H. Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery. Biomolecules. 2025; 15(3):382. https://doi.org/10.3390/biom15030382
Chicago/Turabian StyleYang, Youhui, Yingchen Wang, Zhaoguan Wang, and Hao Qi. 2025. "Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery" Biomolecules 15, no. 3: 382. https://doi.org/10.3390/biom15030382
APA StyleYang, Y., Wang, Y., Wang, Z., & Qi, H. (2025). Efficient Incorporation of DOPA into Proteins Free from Competition with Endogenous Translation Termination Machinery. Biomolecules, 15(3), 382. https://doi.org/10.3390/biom15030382