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. 2021 Apr 23;12:2403. doi: 10.1038/s41467-021-22732-w

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Fluorescence distributions of cells expressing nanobodies comparing the synthetic combinatorial library (pink) and our designed library (green) in two biological replicate experiments as well as a control experiment of a single, well-expressed nanobody clone (Nb174684, purple). The distributions of the designed library are consistently right-shifted compared to the combinatorial library and resemble the control nanobody. b Compared to the combinatorial library, the designed library has almost double the mean expression level (166,193 AU compared to 92,183 AU), nearly half the fraction of poorly expressed nanobodies (of cells expressing nanobodies) (15.4% compared to 25.7% of clones with less than 10,000 AU indicated as a gray bar in panel (a)), and one and a half times the fraction of total cells that express nanobodies (39.6% compared to 25.1%). The thresholds for determining the proportion of total cells expressing nanobodies were found by identifying the local minima on the distributions and are displayed in Supplementary Fig. 10. Values displayed on the bar graphs are means of two biological replicates for the two libraries and one replicate for the control experiment of the single nanobody clone. Replicate measurements are displayed as dots for the two library experiments. c Fluorescence distributions of nanobodies bound to HSA show a rightward shift after screening and selection, indicating a successful enrichment of binders to the target antigen. d On-yeast binding assay of Nb.174684, an HSA binder identified from the designed library with moderate binding affinity. The means of HSA binding (AU) of three replicates are shown and error bars represent standard deviations in measurements at each concentration of HSA. e CDR3 sequence of binder Nb.174684 and the sequences of the nearest neighbors from the natural llama repertoire that was used to train the autoregressive model.