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Primer-Aided Truncation for the Creation of Hybrid Proteins

Title data

Stabel, Robert ; Stüven, Birthe ; Ohlendorf, Robert ; Möglich, Andreas:
Primer-Aided Truncation for the Creation of Hybrid Proteins.
In: Methods in Molecular Biology. Vol. 1596 (2017) . - pp. 287-304.
ISSN 1940-6029
DOI: https://doi.org/10.1007/978-1-4939-6940-1_18

Abstract in another language

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.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry > Chair Biochemistry - Univ.-Prof. Dr. Andreas Möglich
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 570 Life sciences, biology
Date Deposited: 03 May 2017 08:15
Last Modified: 03 May 2017 08:15
URI: https://eref.uni-bayreuth.de/id/eprint/36917