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Methods Mol Biol. 2017;1596:287-304. doi: 10.1007/978-1-4939-6940-1_18.

Primer-Aided Truncation for the Creation of Hybrid Proteins.

Author information

1
Lehrstuhl für Biochemie, Universität Bayreuth, Universitätsstraße 30, 95447, Bayreuth, Germany.
2
Institut für Biologie, Biophysikalische Chemie, Humboldt-Universität zu Berlin, Berlin, Germany.
3
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, MA, USA.
4
Lehrstuhl für Biochemie, Universität Bayreuth, Universitätsstraße 30, 95447, Bayreuth, Germany. andreas.moeglich@uni-bayreuth.de.
5
Institut für Biologie, Biophysikalische Chemie, Humboldt-Universität zu Berlin, Berlin, Germany. andreas.moeglich@uni-bayreuth.de.

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 (primer-aided 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, light-induced 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.

KEYWORDS:

DNA library; Hybrid gene; Light–oxygen–voltage; Protein engineering; Sensor histidine kinase; Sensory photoreceptor; Signal transduction

PMID:
28293894
DOI:
10.1007/978-1-4939-6940-1_18
[Indexed for MEDLINE]

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