Abstract
The ability to sense and process cues about changing environments is fundamental to life. Cells have evolved elaborate signaling pathways in order to respond to both internal and external stimuli appropriately. These pathways combine protein receptors, signal transducers, and effector genes in highly connected networks. The numerous interactions found between signaling proteins are essential to maintain strict regulation and produce a suitable cellular response. As a result, a signaling protein’s activity in isolation can differ greatly from its activity in a native context. This is an important consideration when studying or engineering signaling pathways. Fortunately, the difficulty of studying network interactions is fading thanks to advances in library construction and cell sorting. In this chapter, we describe two methods for generating libraries of mutant proteins that exhibit altered network interactions: whole-gene point mutagenesis and domain shuffling. We then provide a protocol for using fluorescence-activated cell sorting to isolate interesting variants in live cells by focusing on the unicellular eukaryotic model organism Saccharomyces cerevisiae, using as an example recent work that we have done on its G protein-coupled receptor Ste2.
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References
Good MC, Zalatan JG, Lim WA (2011) Scaffold proteins: hubs for controlling the flow of cellular information. Science 332(6030):680–686. doi:10.1126/science.1198701
Peisajovich SG (2012) Evolutionary synthetic biology. ACS Synth Biol 1(6):199–210. doi:10.1021/sb300012g
Romero PA, Arnold FH (2009) Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Biol 10(12):866–876. doi:10.1038/Nrm2805
Leung DW, Chen E, Goeddel DV (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1(1):11–15
Stemmer WPC (1994) DNA shuffling by random fragmentation and reassembly - in-vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91(22):10747–10751. doi:10.1073/pnas.91.22.10747
Eyre-Walker A, Keightley PD (2007) The distribution of fitness effects of new mutations. Nat Rev Genet 8(8):610–618. doi:10.1038/nrg2146
Jackel C, Hilvert D (2010) Biocatalysts by evolution. Curr Opin Biotechnol 21(6):753–759. doi:10.1016/j.copbio.2010.08.008
Tracewell CA, Arnold FH (2009) Directed enzyme evolution: climbing fitness peaks one amino acid at a time. Curr Opin Chem Biol 13(1):3–9. doi:10.1016/j.cbpa.2009.01.017
Szybalski W, Kim SC, Hasan N, Podhajska AJ (1991) Class-IIS restriction enzymes – a review. Gene 100:13–26. doi:10.1016/0378–1119(91)90345-C
Di Roberto RB, Peisajovich SG (2014) The role of domain shuffling in the evolution of signaling networks. J Exp Zool B Mol Dev Evol 322(2):65–72. doi:10.1002/jez.b.22551
Lai A, Sato PM, Peisajovich SG (2015) Evolution of synthetic signaling scaffolds by recombination of modular protein domains. ACS Synth Biol 4(6):714–722. doi:10.1021/sb5003482
Peisajovich SG, Garbarino JE, Wei P, Lim WA (2010) Rapid diversification of cell signaling phenotypes by modular domain recombination. Science 328(5976):368–372. doi:10.1126/science.1182376
Sato PM, Yoganathan K, Jung JH, Peisajovich SG (2014) The robustness of a signaling complex to domain rearrangements facilitates network evolution. PLoS Biol 12(12). doi:10.1371/journal.pbio.1002012
Di Roberto RB, Chang B, Trusina A, Peisajovich SG (2016) Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions. Nat Commun 7:12344. doi:10.1038/ncomms12344
Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96
Acknowledgments
Supporting Grant Information: This work was funded by an NSERC (National Science and Engineering Research Council, Canada) Discovery Grant 418467-2012 (S.G.P), a CFI-ORF (S.G.P.), an Early Research Award from the Province of Ontario (S.G.P.), a Boehringer-Ingelheim Young Researcher Award (S.G.P.), an NSERC Canadian Graduate Scholarship (R.B.D.), and an Ontario Graduate Scholarship (B.S.).
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Di Roberto, R.B., Scott, B.M., Peisajovich, S.G. (2017). Directed Evolution Methods to Rewire Signaling Networks. In: Stein, V. (eds) Synthetic Protein Switches. Methods in Molecular Biology, vol 1596. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6940-1_20
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DOI: https://doi.org/10.1007/978-1-4939-6940-1_20
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