287
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_18, © Springer Science+Business Media LLC 2017
Chapter 18
Primer-Aided Truncation for the Creation of Hybrid
Proteins
Robert Stabel, Birthe Stüven, Robert Ohlendorf, and Andreas Möglich
Abstract
Proteins frequently display modular architecture with several domains and segments connected by linkers.
Proper protein functionality hinges on finely orchestrated interactions among these constituent elements.
The underlying modularity lends itself to the engineering of hybrid proteins via modular rewiring; novel
properties can thus be obtained, provided the linkers connecting the individual elements are conducive to
productive interactions. As a corollary, the process of protein engineering often encompasses the generation and screening of multiple linker variants. To aid these steps, we devised the PATCHY method (primeraided truncation for the creation of hybrid proteins) to readily generate hybrid gene libraries of predefined
composition. We applied PATCHY to the mechanistic characterization of hybrid receptors that possess
blue-light-regulated histidine kinase activity. Comprehensive sampling of linker composition revealed that
catalytic activity and response to light are primarily functions of linker length. Variants with linkers of 7n
residues mostly have light-repressed activity but those with 7n + 1 residues mostly have inverted, lightinduced activity. We further probed linker length in the context of single residue exchanges that also lead
to an inversion of the signal response. As in the original context, activity is only observed for certain periodic linker lengths. Taken together, these results provide mechanistic insight into signaling strategies
employed by sensory photoreceptors and sensor histidine kinases. PATCHY represents an adequate and
facile method to efficiently generate and probe hybrid gene libraries and to thereby identify key determinants for proper function.
Key words DNA library, Hybrid gene, Light–oxygen–voltage, Protein engineering, Sensor histidine
kinase, Sensory photoreceptor, Signal transduction
1 Introduction
Proteins of diverse biological roles consist of multiple modules,
often corresponding to distinct protein domains, that are connected through linker segments [1]. Proper function generally
depends on productive and precisely calibrated interactions
among these modules, as facilitated by the intervening linkers [2].
Depending upon protein context, linkers differ in a number of
parameters including length, sequence, surface charge, structure
and flexibility. Given their eminent role in mediating interactions
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_18, © Springer Science+Business Media LLC 2017
Chapter 18
Primer-Aided Truncation for the Creation of Hybrid
Proteins
Robert Stabel, Birthe Stüven, Robert Ohlendorf, and Andreas Möglich
Abstract
Proteins frequently display modular architecture with several domains and segments connected by linkers.
Proper protein functionality hinges on finely orchestrated interactions among these constituent elements.
The underlying modularity lends itself to the engineering of hybrid proteins via modular rewiring; novel
properties can thus be obtained, provided the linkers connecting the individual elements are conducive to
productive interactions. As a corollary, the process of protein engineering often encompasses the generation and screening of multiple linker variants. To aid these steps, we devised the PATCHY method (primeraided truncation for the creation of hybrid proteins) to readily generate hybrid gene libraries of predefined
composition. We applied PATCHY to the mechanistic characterization of hybrid receptors that possess
blue-light-regulated histidine kinase activity. Comprehensive sampling of linker composition revealed that
catalytic activity and response to light are primarily functions of linker length. Variants with linkers of 7n
residues mostly have light-repressed activity but those with 7n + 1 residues mostly have inverted, lightinduced activity. We further probed linker length in the context of single residue exchanges that also lead
to an inversion of the signal response. As in the original context, activity is only observed for certain periodic linker lengths. Taken together, these results provide mechanistic insight into signaling strategies
employed by sensory photoreceptors and sensor histidine kinases. PATCHY represents an adequate and
facile method to efficiently generate and probe hybrid gene libraries and to thereby identify key determinants for proper function.
Key words DNA library, Hybrid gene, Light–oxygen–voltage, Protein engineering, Sensor histidine
kinase, Sensory photoreceptor, Signal transduction
1 Introduction
Proteins of diverse biological roles consist of multiple modules,
often corresponding to distinct protein domains, that are connected through linker segments [1]. Proper function generally
depends on productive and precisely calibrated interactions
among these modules, as facilitated by the intervening linkers [2].
Depending upon protein context, linkers differ in a number of
parameters including length, sequence, surface charge, structure
and flexibility. Given their eminent role in mediating interactions