A novel framework for engineering protein loops exploring length and compositional variation

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 few avai...

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Autores principales:	Pedro A. G. Tizei, Emma Harris, Shamal Withanage, Marleen Renders, Vitor B. Pinheiro
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c189ef69ca0b404eb475c4a61034aca1
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spelling	oai:doaj.org-article:c189ef69ca0b404eb475c4a61034aca12021-12-02T16:55:54ZA novel framework for engineering protein loops exploring length and compositional variation10.1038/s41598-021-88708-42045-2322https://doaj.org/article/c189ef69ca0b404eb475c4a61034aca12021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-88708-4https://doaj.org/toc/2045-2322Abstract 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 few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion 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.Pedro A. G. TizeiEmma HarrisShamal WithanageMarleen RendersVitor B. PinheiroNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Pedro A. G. Tizei Emma Harris Shamal Withanage Marleen Renders Vitor B. Pinheiro A novel framework for engineering protein loops exploring length and compositional variation
description	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 few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion 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.
format	article
author	Pedro A. G. Tizei Emma Harris Shamal Withanage Marleen Renders Vitor B. Pinheiro
author_facet	Pedro A. G. Tizei Emma Harris Shamal Withanage Marleen Renders Vitor B. Pinheiro
author_sort	Pedro A. G. Tizei
title	A novel framework for engineering protein loops exploring length and compositional variation
title_short	A novel framework for engineering protein loops exploring length and compositional variation
title_full	A novel framework for engineering protein loops exploring length and compositional variation
title_fullStr	A novel framework for engineering protein loops exploring length and compositional variation
title_full_unstemmed	A novel framework for engineering protein loops exploring length and compositional variation
title_sort	novel framework for engineering protein loops exploring length and compositional variation
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/c189ef69ca0b404eb475c4a61034aca1
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A novel framework for engineering protein loops exploring length and compositional variation

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