Peptide barcoding for multiplex evaluation of affinities of nanobody libraries

Background Binding proteins such as antibodies are useful for basic science, diagnosis, and therapy. Nanobodies also promising binding that achieve comparable affinities specificities to conventional antibodies, despite comprising only a single 15 kDa variable domain. The generation of functional nanobodies typically involves phage, yeast, or ribosome display, which allows screening billions simultaneously. However, display technologies have some drawbacks, one is the immobilization effect. on cell surface necessary establish genotype–phenotype linkages, but this can affect nanobody functions. Therefore, we developed peptide barcoding, technology with evaluate free low bias [1]. Here, applied alanine scanning anti-GFP enabled multiplex evaluation nanobodies. Method We used GFP model antigen antibody. was produced by E. coli BL21 (DE3) strain. Anti-GFP were P. pastoris GS115 To construct alanine-scanning library, substituted each non-alanine residue prepared 106 kinds Each attached genetically encoded unique barcode. nanobody–peptide barcode fusion (barcodebodies) in pot pastoris, digested trypsin, LC-MS/MS confirm detectability barcodes. barcodes, study, derived from yeast SRM Atlas where tens thousands proteotypic peptides registered [2]. barcodebody library mixed separated size-exclusion chromatography depending affinity. fractions containing high-affinity barcodebodies low-affinity trypsin LC-MS/MS. Results & Discussion detected more than 95% In fraction barcodebodies, simultaneously identified five other measured affinity these plasmon resonance (SPR) confirmed their decrease affinities. These results demonstrated practicality utility barcoding one-pot non-immobilized suggested could be high-throughput screening. [1] Miyamoto et al., PLOS ONE 14(4): e0215993, 2019 [2] Picotti Nature 494(7436), 266-270, 2013