期刊
SCIENTIFIC REPORTS
卷 11, 期 1, 页码 -出版社
NATURE RESEARCH
DOI: 10.1038/s41598-021-88708-4
关键词
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资金
- CAPES foundation [BEX 8985-13-8]
- European Research Council [336936]
- BBSRC [BB/K018132/1]
- European Research Council (ERC) [336936] Funding Source: European Research Council (ERC)
- BBSRC [BB/K018132/1] Funding Source: UKRI
Insertions and deletions (indels) impact enzyme function, biophysical properties, and substrate specificity, yet remain underutilized in protein engineering. The InDel assembly and analysis platforms offer an efficient approach for systematically generating and analyzing libraries with varying sequence composition and length, enabling the engineering of protein loops with essential functional parameters.
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 beta -lactamase Omega -loop, involved in substrate specificity, identifying multiple novel extended spectrum beta -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.
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