Author:
Matsuzaki Yusei,Aoki Wataru,Miyazaki Takumi,Aburaya Shunsuke,Ohtani Yuta,Kajiwara Kaho,Koike Naoki,Minakuchi Hiroyoshi,Miura Natsuko,Kadonosono Tetsuya,Ueda Mitsuyoshi
Abstract
AbstractOptimisation of protein binders relies on laborious screening processes. Investigation of sequence–function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence–function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody–antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence–function relationships.
Funder
JST FOREST
Core Research for Evolutional Science and Technology
JST COI-NEXT
Publisher
Springer Science and Business Media LLC
Cited by
1 articles.
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