Large Scale Synthetic Site Saturation GPCR Libraries Reveal Novel Mutations That Alter Glucose Signaling.

Autor: Öling D; Discovery Biology, Discovery Sciences, Innovative Medicines and Early Development Biotech Unit , AstraZeneca R&D , 431 50 Mölndal , Sweden., Lawenius L; Discovery Biology, Discovery Sciences, Innovative Medicines and Early Development Biotech Unit , AstraZeneca R&D , 431 50 Mölndal , Sweden., Shaw W; Department of Bioengineering , Imperial College London , London SW7 2AZ , U.K.; Centre for Synthetic Biology , Imperial College London , London SW7 2AZ , U.K., Clark S; Twist Bioscience , San Francisco , California 94158 , United States., Kettleborough R; Twist Bioscience , San Francisco , California 94158 , United States., Ellis T; Department of Bioengineering , Imperial College London , London SW7 2AZ , U.K.; Centre for Synthetic Biology , Imperial College London , London SW7 2AZ , U.K., Larsson N; Discovery Biology, Discovery Sciences, Innovative Medicines and Early Development Biotech Unit , AstraZeneca R&D , 431 50 Mölndal , Sweden., Wigglesworth M; Hit Identification, Discovery Sciences, Innovative Medicines and Early Development Biotech Unit , AstraZeneca R&D , Macclesfield SK10 2NA , U.K.
Jazyk: angličtina
Zdroj: ACS synthetic biology [ACS Synth Biol] 2018 Sep 21; Vol. 7 (9), pp. 2317-2321. Date of Electronic Publication: 2018 Sep 12.
DOI: 10.1021/acssynbio.8b00118
Abstrakt: Site saturation mutagenesis (SSM) is a powerful mutagenesis strategy for protein engineering and directed evolution experiments. However, limiting factors using this method are either biased representation of variants, or limiting library size. To overcome these hurdles, we generated large scale targeted synthetic SSM libraries using massively parallel oligonucleotide synthesis and benchmarked this against an error-prone (epPCR) library. The yeast glucose activated GPCR-Gpr1 was chosen as a prototype to evolve novel glucose sensors. We demonstrate superior variant representation and several unique hits in the synthetic library compared to the PCR generated library. Application of this mutational approach further builds the possibilities of synthetic biology in tuning of a response to known ligands and in generating biosensors for novel ligands.
Databáze: MEDLINE
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