321
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_20, © Springer Science+Business Media LLC 2017
Chapter 20
Directed Evolution Methods to Rewire Signaling Networks
Raphaël B. Di Roberto, Benjamin M. Scott, and Sergio G. Peisajovich
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.
Key words Signaling pathway, Protein network, Directed evolution, Domain shuffling, Random
mutagenesis, Error-prone PCR, Fluorescence-activated cell sorting
1 Introduction
Cells must constantly respond to changes in their environment to
fulfill their role or simply to survive. It is the function of signaling
pathways to detect these changes and to decide on an appropriate
response. These pathways are composed of a variety of proteins
and small molecules interacting together and forming highly connected networks. Signaling proteins include extracellular or intracellular receptors, small molecules, modifiers (kinases, methylases,
proteases, etc.) and transcription factors, among others.
Due to their complex roles in signal transmission and regulation, signaling networks are especially rich in protein–protein
interactions. These interactions encompass simple binding, or scaffolding, which ensures that effectors and their targets are in close
proximity, as well as more complex regulatory interactions that can
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_20, © Springer Science+Business Media LLC 2017
Chapter 20
Directed Evolution Methods to Rewire Signaling Networks
Raphaël B. Di Roberto, Benjamin M. Scott, and Sergio G. Peisajovich
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.
Key words Signaling pathway, Protein network, Directed evolution, Domain shuffling, Random
mutagenesis, Error-prone PCR, Fluorescence-activated cell sorting
1 Introduction
Cells must constantly respond to changes in their environment to
fulfill their role or simply to survive. It is the function of signaling
pathways to detect these changes and to decide on an appropriate
response. These pathways are composed of a variety of proteins
and small molecules interacting together and forming highly connected networks. Signaling proteins include extracellular or intracellular receptors, small molecules, modifiers (kinases, methylases,
proteases, etc.) and transcription factors, among others.
Due to their complex roles in signal transmission and regulation, signaling networks are especially rich in protein–protein
interactions. These interactions encompass simple binding, or scaffolding, which ensures that effectors and their targets are in close
proximity, as well as more complex regulatory interactions that can