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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Xiao H, Schultz PG (2016) At the Interface of chemical and biological synthesis: an expanded genetic code. Cold Spring Harb Perspect Biol 8(9):a023945
Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444
Chin JW (2014) Expanding and reprogramming the genetic code of cells and animals. Annu Rev Biochem 83:379–408
Weissman KJ (2015) The structural biology of biosynthetic megaenzymes. Nat Chem Biol 11:660–670
Xu W, Qiao K, Tang Y (2013) Structural analysis of protein-protein interactions in type I polyketide synthases. Crit Rev Biochem Mol Biol 48:98–122
Ongley SE, Bian X, Neilan BA, MĂ¼ller R (2013) Recent advances in the heterologous expression of microbial natural product biosynthetic pathways. Nat Prod Rep 30:1121–1138
Zhang H, Boghigian BA, Armando J, Pfeifer BA (2011) Methods and options for heterologous production of complex natural products. Nat Prod Rep 28:125–151
Baltz RH (2010) Streptomyces and Saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters. J Ind Microbiol Biotechnol 37:759−772
Chin J, Santoro S, Martin A, King D, Wang L, Schultz P (2002) Addition of p-Azido-L-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc 124:9026–9027
Chin JW, Martin AB, King DS, Wang L, Schultz PG (2002) Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl Acad Sci U S A 99:11020–11024
Pédelacq J, Cabantous S, Tran T, Terwilliger T, Waldo G (2005) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88
He J, Van Treeck B, Nguyen HB, Melançon CE III (2016) Development of an unnatural amino acid incorporation system in the Actinobacterial natural product producer Streptomyces venezuelae ATCC 15439. ACS Synth Biol 5:125–132
Melançon C, Liu H (2007) Engineered biosynthesis of macrolide derivatives bearing the non-natural Deoxysugars 4-epi-D-Mycaminose and 3-N-Monomethylamino-3-Deoxy-D-Fucose. J Am Chem Soc 129:4896–4897
Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. The John Innes Foundation, Norwich
Mahmood T, Yang P-C (2012) Western blot: technique, theory, and trouble shooting. North American J Med Sci 4:429–434
Bornhorst JA, Falke JJ (2000) Purification of proteins using Polyhistidine affinity tags. Methods Enzymol 326:245–254
Acknowledgments
We gratefully acknowledge Peter G. Schultz of the Scripps Research Institute for providing the plasmid harboring MjtRNATyr CUA and Hung-wen Liu at The University of Texas at Austin for providing S. venezuelae ATCC 15439. This work is supported by NIH NM-INBRE grant P20 GM103451 (C.E.M.) and by the University of New Mexico.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
He, J., Melançon, C.E. (2018). Genetic Incorporation of Unnatural Amino Acids into Proteins of Interest in Streptomyces venezuelae ATCC 15439. In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7574-7_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7573-0
Online ISBN: 978-1-4939-7574-7
eBook Packages: Springer Protocols