This study is about 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.
Fig: Producing length and compositional variation with InDel assembly. (a) At each assembly cycle, dsDNA templates bound to the paramagnetic beads are restricted with SapI (a type IIs endonuclease), building blocks annealed and ligated. After ligation, the cycle can be restarted. Compositional variation is achieved primarily by combining pools of different building blocks. (b) Denaturing gel electrophoresis of fluorescently labelled template across the different steps of the cycle show that restriction digestion and ligation are not carried out to completion in any step, underpinning the length variation of the resulting libraries. (c) Sequence of a building block. A long double-stranded region is used to stabilize building block annealing and ensure efficient endonuclease activity.
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.
Tizei, P.A.G., Harris, E., Withanage, S. et al. A novel framework for engineering protein loops exploring length and compositional variation. Sci Rep 11, 9134 (2021). https://doi.org/10.1038/s41598-021-88708-4