Abstract
Site-specific, genetic incorporation of unnatural amino acids (UAAs) into proteins in living cells using engineered orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs is a powerful tool for studying and manipulating protein structure and function. To date, UAA incorporation systems have been developed for several bacterial and eukaryotic model hosts. Due to the importance of Streptomyces as prolific producers of bioactive natural products and as model hosts for natural product biosynthesis and bioengineering studies, we have developed systems for the incorporation of the UAAs p-iodo-L-phenylalanine (pIPhe) and p-azido-L-phenylalanine (pAzPhe) into green fluorescent protein (GFP) in Streptomyces venezuelae ATCC 15439. Here, we describe the procedure for using this system to site-specifically incorporate pIPhe or pAzPhe into proteins of interest in S. venezuelae. The modular design of plasmids harboring UAA incorporation systems enables use of other aaRS or aaRS/tRNA pairs for the incorporation of other UAAs; and the vector backbone used allows the system to be transferred to diverse Streptomyces species via both protoplast transformation and conjugation.
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