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Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Number of Authors: 3
2017 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 30, no 8, 543-549 p.Article in journal (Refereed) Published
Abstract [en]

Exploring the vicinity around a locus of a protein in sequence space may identify homologs with enhanced properties, which could become valuable in biotechnical and other applications. A rational approach to this pursuit is the use of 'infologs', i.e. synthetic sequences with specific substitutions capturing maximal sequence information derived from the evolutionary history of the protein family. Ninety-five such infolog genes of poplar glutathione transferase were synthesized and expressed in Escherichia coli, and the catalytic activities of the proteins determined with alternative substrates. Sequence-activity relationships derived from the infologs were used to design a second set of 47 infologs in which 90% of the members exceeded wild-type properties. Two mutants, C2 (V55I/E95D/D108E/A160V) and G5 (F13L/C70A/G122E), were further functionally characterized. The activities of the infologs with the alternative substrates 1-chloro-2,4-dinitrobenzene and phenethyl isothiocyanate, subject to different chemistries, were positively correlated, indicating that the examined mutations were affecting the overall catalytic competence without major shift in substrate discrimination. By contrast, the enhanced protein expressivity observed in many of the mutants were not similarly correlated with the activities. In conclusion, small libraries of well-defined infologs can be used to systematically explore sequence space to optimize proteins in multidimensional functional space.

Place, publisher, year, edition, pages
2017. Vol. 30, no 8, 543-549 p.
Keyword [en]
alternative substrates, directed protein evolution, enhanced activities, glutathione transferase, infologs
National Category
Biological Sciences Environmental Biotechnology
Identifiers
URN: urn:nbn:se:su:diva-148911DOI: 10.1093/protein/gzx045ISI: 000412298600002PubMedID: 28967959OAI: oai:DiVA.org:su-148911DiVA: diva2:1157246
Available from: 2017-11-15 Created: 2017-11-15 Last updated: 2017-11-15Bibliographically approved

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