Site-directed cross-linking with KLK proteases from prostate
Final Report Abstract
This project focused on improving and designing new orthogonal components for protein translation with "clickable" noncanonical amino acids (ncAAs) to create molecular tools for bioconjugation. Human kallikrein-related peptidases (KLKs), involved in fertilization and prostate cancer, were selected as the model proteins. The goal was to apply Selective Pressure Incorporation (SPI) and Stop Codon Suppression (SCS) methods in KLK studies. While SPI is well-established, especially with "clickable" ncAAs, the orthogonal translation system achieved a breakthrough by efficiently incorporating more than 20 ncAAs into model proteins. These ncAAs, containing small bioorthogonal tags, are ideal for various click chemistry applications. Additionally, system efficiency was significantly improved by introducing solubility tags to enhance enzyme folding and utilizing translational components from psychrophilic (cold-adapted) organisms for enhanced performance ("cold" orthogonal translation).
Publications
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Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags. Frontiers in Bioengineering and Biotechnology.
Koch, Nikolaj G.; Baumann, Tobias & Budisa, Nediljko
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Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains. International Journal of Molecular Sciences, 22(20), 11194.
Koch, Nikolaj G.; Goettig, Peter; Rappsilber, Juri & Budisa, Nediljko
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Pyrrolysyl-tRNA-Synthetase: Methanogenese und Gencode-Erweiterung. BIOspektrum, 27(6), 616-619.
Koch, Nikolaj Georg & Budisa, Nediljko
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Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various Noncanonical Amino Acids. Methods in Molecular Biology, 3-19. Springer US.
Koch, Nikolaj G. & Budisa, Nediljko
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Non-Canonical Amino Acids in Analyses of Protease Structure and Function. International Journal of Molecular Sciences, 24(18), 14035.
Goettig, Peter; Koch, Nikolaj G. & Budisa, Nediljko
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“Cold” Orthogonal Translation: A Psychrophilic Pyrrolysyl-tRNA Synthetase Boosts Genetic Code Expansion in E. coli. openRxiv.
Koch, Nikolaj G.; Goettig, Peter; Nash, Michael A.; Rappsilber, Juri & Budisa, Nediljko
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Evolution of Pyrrolysyl-tRNA Synthetase: From Methanogenesis to Genetic Code Expansion. Chemical Reviews, 124(16), 9580-9608.
Koch, Nikolaj G. & Budisa, Nediljko
