Efficient Incorporation of Clickable Unnatural Amino Acids Enables Rapid and Biocompatible Labeling of Proteins in Vitro and in Bacteria |
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Authors: | Dagan Hadar Sigal Gelkop Livne Vaserman Dr Miriam Amiram |
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Affiliation: | 1. Avram and Stella Goldstein-Goren 2. Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva, 8410501 Israel |
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Abstract: | Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment – typically at a single site or at a few sites per protein – of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l -phenylalanine (pPR), with ~30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production – using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain – of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling. |
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Keywords: | copper-catalyzed azide-alkyne cycloadditions elastin-like polypeptides protein modifications unnatural amino acids |
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