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• W3101430275 abstract "Abstract Insertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with no available platforms capable of systematically generating or analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 β-lactamase Ω-loop, involved in substrate specificity, identifying multiple novel extended spectrum β-lactamases with loops of modified length and composition areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters." @default.
• W3101430275 created "2020-11-23" @default.
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• W3101430275 date "2017-04-15" @default.
• W3101430275 modified "2023-10-14" @default.
• W3101430275 title "InDel assembly: A novel framework for engineering protein loops through length and compositional variation" @default.
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• W3101430275 doi "https://doi.org/10.1101/127829" @default.
• W3101430275 hasPublicationYear "2017" @default.
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