Yeast surface display platform for rapid discovery of conformationally selective nanobodies

Transcription

1 SUPPLEMENTARY Technical INFORMATION Report In the format provided by the authors and unedited. Yeast surface display platform for rapid discovery of conformationally selective nanobodies Conor McMahon 1, Alexander S. Baier 1, Roberta Pascolutti 1, Marcin Wegrecki 2, Sanduo Zheng 1, Janice X. Ong 1, Sarah C. Erlandson 1, Daniel Hilger 3, Søren G. F. Rasmussen 2, Aaron M. Ring 4, Aashish Manglik 5,6 * and Andrew C. Kruse 1 * 1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA. 2 Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark. 3 Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA. 4 Department of Immunobiology, Yale School of Medicine, New Haven, CT, USA. 5 Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA. 6 Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, CA, USA. * Aashish.Manglik@ucsf.edu; Andrew_Kruse@hms.harvard.edu Nature Structural & Molecular Biology Nature America Inc., part of Springer Nature. All rights reserved.

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3 Supplementary Figure 1 Biochemical validation of nanobody clones (A K) Randomly chosen nanobodies were expressed and purified from E. coli, then analyzed by size exclusion chromatography to assess monodispersity. (L) SDS-PAGE analysis of nanobody purity following one-step nickel affinity purification.

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5 Supplementary Figure 2 Design of display system (A) The display system was engineered using the high affinity SIRPα variant CV1 as a test protein, and its ligand CD47 ectodomain as the staining reagent. A biotin tag is schematized as a glowing red circle. (B) Length of the stalk region determines accessibility of a displayed protein as a function of molecular weight. (C) Analytical flow cytometry plots showing length dependence for two staining reagents: CD47 biotin and α-ha antibody. The 649 amino acid long stalk was used in all nanobody display experiments.

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7 Supplementary Figure 3 Analysis of HSA-targeted nanobodies (A) Library design was assessed by monitoring the change in amino acid frequency in CDR3 throughout selection rounds with HSA as the antigen. Few changes were observed, with the only notable trend a modest increase in basic residue frequency and a decline in acidic residue frequency. (B) Assessment of Nb.b201 binding to human serum albumin by surface plasmon resonance, comparison with mouse serum albumin which shows no detectable binding. (C) 2Fo-Fc composite omit map contoured at 1.5 σ for antigen bound Nb.b201. The structure of both bound (yellow) and free (gray) forms of the nanobody are shown, highlighting structural divergence. (D) 2Fo-Fc composite omit map contoured at 1.5 σ for free Nb.b201.

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9 Supplementary Figure 4 Discovery of nanobodies with nonpurified antigen (A) Conditioned medium containing adiponectin (left lane) was used for selection of nanobodies. It shows a complex mixture of proteins as assessed by SDS-PAGE. For reference, purified adiponectin is shown in the right lane. Adiponectin exists as a mix of 16-mers, hexamers, and trimers. (B) Schematic of selection process. Fluorescent anti-flag antibody was used to specifically mark those yeast cells that display adiponectinbinding nanobodies. (C) Flow cytometry analysis of final clone pool, showing that the library was highly enriched in adiponectin-binding clones. (D) Sequences of five selected clones showed highly diverse CDR3 sequence composition and length. (E) Binding assessed using in vitro pull-down with purified adiponectin globular domain. (F) Binding to adiponectin was further confirmed in vitro using surface plasmon resonance. Kinetic fit is shown for clone Nb.AQ103.

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11 Supplementary Figure 5 Affinity of β2ar-binding nanobodies On-yeast titration to estimate affinity of β2ar binding nanobodies. EC50 values are summarized in the lower right. Bottom panel shows measurement of conformational selectivity for selected clones as assessed by flow cytometry.

12 Nanobody Sequence Notes Naive library biochemical assessment Nb.BV008 QVQLQESGGGLVQAGGSLRLSCAASGYISTTFA MGWYRQAPGKERELVAAIDKGSSTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAR YYDYPLDGTYHIYWGQGTQVTVSS Monodisperse Nb.BV009 QVQLQESGGGLVQAGGSLRLSCAASGNIFAKP YMGWYRQAPGKERELVATITYGSNTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA AAVTYWLYTQAYWYWGQGTQVTVSS Poorly expressed and polydisperse Nb.BV018 QVQLQESGGGLVQAGGSLRLSCAASGNIFRRG WMGWYRQAPGKERELVASIAGGTNTNYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA NTAYWNDYYYWGQGTQVTVSS Monodisperse Nb.BV025 QVQLQESGGGLVQAGGSLRLSCAASGSISLYAE MGWYRQAPGKERELVAGITRGTTTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAD YVQGKSRVIPHYYWGQGTQVTVSS Monodisperse Nb.BV032 QVQLQESGGGLVQAGGSLRLSCAASGSISDSYL MGWYRQAPGKERELVASISRGGITNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVV STWLWDLEYWGQGTQVTVSS Mostly monodisperse Nb.BV035 QVQLQESGGGLVQAGGSLRLSCAASGTISNYY YMGWYRQAPGKEREFVASINYGASTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV YTYHGTYYFYWGQGTQVTVSS Monodisperse, low expression Nb.BV045 QVQLQESGGGLVQAGGSLRLSCAASGYISPAS VMGWYRQAPGKEREFVASIDFGGNTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA YVDQDIGTAGYHAYWGQGTQVTVSS Monodisperse Nb.BV047 QVQLQESGGGLVQAGGSLRLSCAASGNIFSGG GMGWYRQAPGKERELVAAIGSGGNTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVI WADLRSFFYWGQGTQVTVSS Somewhat polydisperse Nb.BV049 QVQLQESGGGLVQAGGSLRLSCAASGNIFRGV AMGWYRQAPGKERELVAAITLGTSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA WSTYVPGDPYTYTYWGQGTQVTVSS Monodisperse Nb.BV052 QVQLQESGGGLVQAGGSLRLSCAASGTISLTAT MGWYRQAPGKERELVAGISSGTITNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVG NYDRRITWYGFGYWGQGTQVTVSS Monodisperse Nb.BV056 QVQLQESGGGLVQAGGSLRLSCAASGTISRTFV MGWYRQAPGKEREFVASIAIGANTNYADSVKG Monodisperse

13 Human Serum Albumin Nb.b201 Adiponectin Nb.AQ100 Nb.AQ101 Nb.AQ102 Nb.AQ103 Nb.AQ104 A 2A R Nb.AD101 Nb.AD102 β2-adrenergic receptor Nb.c200 Nb.c201 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVY LYATDTPDRDFAYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGYISDAY YMGWYRQAPGKEREFVATITHGTNTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV LETRSYSFRYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGTIFRVSF MGWYRQAPGKEREFVASITYGGITNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYI LVSVDGAYYYRYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGYIFVYAY MGWYRQAPGKEREFVAAIDVGTTTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVV VAPQYFYWSTADRRYLYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGSIFRVA WMGWYRQAPGKERELVASINGGATTNYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV PSTLGGDFYYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGSIFAAAY MGWYRQAPGKEREFVAAISDGSNTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVIR WNYTYFRYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGYIFPSAV MGWYRQAPGKERELVATITYGGSTYYADSVKG RFTISRDNAKNTVYLQMNLKPEDTAVYYCAAGIF TFIKGLVDPGYSLAYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGTISPLPA MGWYRQAPGKEREFVAGIDTGAITNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVF PAAYDYYERYYTYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGSIFSILS MGWYRQAPGKERELVAGITDGTTTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVH AVPDEYTFPSSYDYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGNISWVY GMGWYRQAPGKEREFVAAINYGSITNYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV AYATYAVFDPYFWYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGSISYYS SMGWYRQAPGKERELVASIDTGTSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV YTSWYSPYIYWGQGTQVTVSS K D = 431 ± 12 nm K D = 2.3 ± 0.38 μm N/A N/A K D = 510 ± 28 nm N/A May have reduced binding on freeze/thaw K D = 78 nm (SPR) K D = 147 nm (SPR)

14 Nb.c202 Nb.c203 Nb.c204 Nb.c205 Nb.c206 Nb.c207 Nb.c208 Nb.c209 Nb.c210 Nb.c211 Nb.c212 QVQLQESGGGLVQAGGSLRLSCAASGSISYDG HMGWYRQAPGKERELVAGIDFGGITYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA DPGVQSSSLETFFTFFLYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGTIFYDTA MGWYRQAPGKERELVATIDDGTSTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAY SHTYTIFPWTLFYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGTIFGTVL MGWYRQAPGKEREFVAGISVGGSTNYADSVKG RFTISRDNDTVYLQMNSLKPEDTAVYYCAVFDIS FLVFPWSHDYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGTISYYY GMGWYRQAPGKERELVAAIADGSSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV FELRGFLWDWYYTYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGYIFITVP MGWYRQAPGKEREFVASIDYGTTTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVH DINSYLSLEGFWVDHVYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGNISYWY YMGWYRQAPGKEREFVAGIDGGSNTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA LAWRDSYSDPWFYYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGNIFSYY YMGWYRQAPGKERELVAAIGVGANTNYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV TNPWYWIGVPAADSYYDYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLRCAASGSISVYFF MGWYRQAPGKERELVASINPGGNTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVP VLGYATAINYHWYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGSIFRQD LMGWYRQAPGKEREFVAGIGQGTSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV ARQLWDYYYDTHYYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGSIFGDV DMGWYRQAPGKEREFVAAIAYGGSTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HSWVTDYYPFEHYYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGYIFLRLT MGWYRQAPGKERELVATITTGGNTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAP VPIFRSLGTFFGPNHWYWGQGTQVTVSS K D = 44 nm (SPR) K D = 151 nm (SPR) EC 50 on yeast: 49 nm EC 50 on yeast: 21 nm EC 50 on yeast: 77 nm EC 50 on yeast: 104 nm EC 50 on yeast: 55 nm EC 50 on yeast: 24 nm EC 50 on yeast: 2.1 μm EC 50 on yeast: 62 nm EC 50 on yeast: 20 nm

15 General primers pydsfor1: GAAGGTGTTCAATTGGACAAGAGAGAAGCTGACGCAcagg tgcagctgcaggaaagcggc pydsrev1: TACTGATGCTTCTGTAGAGGGTGAGGATGTTTGAGCGTA ATCTGGAACATCGTATGGGTAGGATCCgctgctcacggtcacctg ggtgcc pydsfor2: ACCACCATCGCTTCTATCGCTGCTAAGGAAGAAGGTGTT CAATTGGACAAGAGAGAAGC pydsrev2: TACTGATGCTTCTGTAGAGGGTGAGG pydsfor3: ACCACCATCGCTTCTATCGCTGCTAAG Batch1For: TCGAAGGGACAAGAGAGAAGCTGACGCA Batch1Rev: CTTCGACTGGAACATCGTATGGGTAGGATCC Batch2For: ACCTGAGGACAAGAGAGAAGCTGACGCA Batch2Rev: TCAGGTCTGGAACATCGTATGGGTAGGATCC Batch3For: GCAATCGGACAAGAGAGAAGCTGACGCA Batch3Rev: GATTGCCTGGAACATCGTATGGGTAGGATCC Rd1For: GACTACGGACAAGAGAGAAGCTGACGCA Rd1Rev: GTAGTCCTGGAACATCGTATGGGTAGGATCC Rd2For: CGAGTTGGACAAGAGAGAAGCTGACGCA Rd2Rev: AACTCGCTGGAACATCGTATGGGTAGGATCC Rd3For: GCTAGAGGACAAGAGAGAAGCTGACGCA Rd3Rev: TCTAGCCTGGAACATCGTATGGGTAGGATCC Rd4For: CTCTACGGACAAGAGAGAAGCTGACGCA Rd4Rev: GTAGAGCTGGAACATCGTATGGGTAGGATCC pet26b_nblib_ga_for: CTCCTCGCTGCCCAGCCGGCGATGGCCcaggtgcagctgcagg

16 aaag pet26b_nblib_ga_rev: CGGATCTCAGTGGTGGTGGTGGTGGTGgctgctcacggtcacct gg Nanobody library construction primers P1_for: caggtgcagctgcaggaaagcggcggcggcctggtgcaggcgggcggcag P2_rev: gccgctcgccgcgcagctcaggcgcaggctgccgcccgcctgc Keck Biotechnology Resource Laboratory Keck Biotechnology Resource Laboratory P3_for: cgcggcgagcggcwmtatttyt####atgggctggtatcgccagg Keck Biotechnology Resource Laboratory P4_rev: ttcgcgttctttgcccggcgcctggcgataccagcccat P5_for: ccgggcaaagaacgcgaayttgttgccrstattrvt#ggtrstantacc WATtatgcggatagcgtgaaaggcc P6_rev: gtttttcgcgttatcgcggctaatggtaaagcggcctttcacgctatccgcata P7_for: agccgcgataacgcgaaaaacaccgtgtatctgcagatgaacagcctgaaacc P8_rev: cgcgcaataatacaccgcggtatcttccggtttcaggctgttcatctgcaga P9a_for: ccgcggtgtattattgcgcggyt#######ywt#tattggggccagggcacc P9b_for: ccgcggtgtattattgcgcggyt###########ywt#tattggggccagggc acc P9c_for: Keck Biotechnology Resource Laboratory Keck Biotechnology Resource Laboratory Keck Biotechnology Resource Laboratory Keck Biotechnology Resource Laboratory Keck Biotechnology Resource Laboratory Keck Biotechnology Resource Laboratory Keck Biotechnology Resource Laboratory Keck

17 ccgcggtgtattattgcgcggyt###############ywt#tattggggcca gggcacc P10_rev: gctgctcacggtcacctgggtgccctggccccaata Biotechnology Resource Laboratory Keck Biotechnology Resource Laboratory

18 Nanobody Library Selection by MACS Introduction We have written the protocol below with the goal of making steps of in vitro nanobody selection as clear and broadly applicable as possible, but it is important to recognize that any nanobody discovery effort can have unique requirements. Please view this protocol as a starting point for designing your experiment, rather than the final word on how to do the selection. Variations of this approach have been successful for isolating nanobodies for almost every antigen tested, with the notable exception of unstructured peptides. The key challenge in any selection experiment is not to avoid losing active clones, but rather to efficiently clear out the millions of nanobodies without the desired activity. It is absolutely essential to diligently remove nanobodies that bind to secondary antibodies, microbeads, or protein tags. If you allow the selection process to find a shortcut, it will. Taking these considerations into account, selection with most antigens will yield nanobodies with affinities ranging from 10 nm to 1 μm. Recipes and catalog numbers listed in appendix for items in single quotes Library recovery and expansion 1. Place frozen aliquots of yeast nanobody library (NbLib) at 30 C to thaw, such that recoverable yeast exceed library diversity at least five-fold. For the full naïve library, a total of viable cells are required. 2. Recover yeast in 1 L of Yglc4.5 Trp, shaking at 230 RPM, 30 C, overnight. You may want to take a small sample of the library cells immediately after resuspension and plate on YPD agar to measure the diversity. 3. Expand to 3 L of medium and allow yeast to grow to stationary phase. Approximately 48 hours, OD is usually between 10 and Measure OD 600 of yeast to calculate density (OD 600 of yeast) 5. Spin down cultures at 3500 g for 5 minutes and resuspend in Yglc4.5 Trp supplemented with 10% DMSO, such that final density is cells per ml. Aliquot to 2 ml cryovials and freeze in a cell freezing chamber at -80 C (Thermofisher Scientific cat# ). Each vial should contain enough viable cells upon recovery to reconstitute a full library.

19 Induction of nanobody expression Nanobody expression is under the control of the GAL1 promoter, so nanobodies are only produced on the cell surface when the yeast are grown in galactose-containing medium. 1. Induce nanobody expression of NbLib by dilution of 10 x library diversity into Trp +galactose medium followed by shaking for 48 hours, 25 C, 220 rpm. Use at least yeast cells for the inoculum to ensure no clones are lost in passaging. (We find best expression levels to be between 48 and 72 hours.) Note about sterility: It is essential to maintain a sterile environment throughout your selection. Unlike standard yeast work, your yeast cells will be passaged multiple times over the course of weeks, increasing the possibility of contamination. We recommend sterile filter tips and freshly sterilized media for every selection. Wipe down your hands, bench, and pipettes with ethanol often during the selection process. A sterile hood could be used as well, but usually a regular lab bench is fine so long as reasonable care is taken. Nanobody expression test Before any selection it s a good idea to confirm that nanobody expression was effectively induced. We check this with a quick analytical flow cytometry assay as described below. 1. Measure cell density of induced NbLib yeast (1 OD x 10 7 cells/ml) 2. Add 500 μl of selection buffer to two microcentrifuge tubes 3. Pipette approximately induced yeast into each microcentrifuge tube and centrifuge for 1 min at 3500 g, 4 C 4. Aspirate supernatant and resuspend pellet in 100 μl selection buffer 5. To one tube add ~0.5 μg of anti-ha antibody labeled with AlexaFluor647 or AlexaFluor488 (to check for the percentage of cells expressing nanobody) and 1 μm final concentration of your protein labeled with a spectroscopically distinct dye (to check if your antigen binds nonspecifically to yeast). Leave the other tube unstained as a control 6. Rock both tubes at 4 C for 15 minutes 7. Spin down cells for 1 minute at 3500 g, 4 C 8. Aspirate supernatant and resuspend cells in 500 μl of selection buffer 9. Spin down cells as before and aspirate supernatant 10. Resuspend cells in 100 μl of selection buffer and assess nanobody expression level on flow cytometer using unstained sample to set gates

20 Note: The maximum number of expressing cells from the naïve library is ~25%. The typical number of expressing cells is ~15-20%. If expression is on the low end (~8-12%) consider increasing the number of cells used for nanobody selection to compensate. Example flow cytometer yeast gating: Nanobody Selection The selection process below is at the heart of identifying clones that bind to your target protein. This protocol describes how to separate antigen binding nanobody clones from non-binders using magnetic cell sorting in two major steps: a pre-clear or negative selection step, followed by a positive selection step. These are usually done in series on the same day. The pre-clear step serves to deplete the library of any nanobodies that bind to things like secondary antibodies, magnetic beads, etc. The positive selection step isolates clones that bind to your antigen. After selection, you can expand your enriched cells and repeat the process until binders are highly enriched. 11. Spin down a suitable number of cells to have at least 10-fold over library diversity. For the naïve library (NbLib) you will need cells. After one or more rounds of selection a smaller number of cells may be used based on estimated diversity. We recommend always using at least cells, since smaller numbers are difficult to manipulate. Perform centrifugation for 3 minutes at 3500 g, 4 C 12. Aspirate media and resuspend yeast via pipetting in 10 ml of chilled selection buffer 13. Spin down yeast for 3 minutes at 3500 g, 4 C and aspirate supernatant 14. Resuspend yeast in 4.5 ml of selection buffer

21 15. Begin preclear to remove off-target binders from the naïve library by adding 500 μl of the Miltenyi anti-fluorophore beads to the resuspended yeast (If using a secondary for selection, add that to the preclearing reaction as well. ~200 nm is typically sufficient to remove secondary binders) 16. Rock/Rotate slowly at 4 C for 40 minutes 17. Approximately 20 minutes prior to the conclusion of incubation, place an LD column on the Miltenyi MACS magnet. Put a 15 ml sterile falcon under the column and place a 50mL falcon cap over the top of the column to maintain sterility. Add 5 ml of selection buffer to the LD column to allow it to equilibrate before the next step. It will take ~20 minutes to flow through completely 18. Spin down yeast for 3 minutes at 3500 g at 4 C and aspirate buffer and unbound beads 19. Resuspend by pipetting in 5 ml of selection buffer. 20. Flow yeast over equilibrated LD column, collect flowthrough in new sterile 15mL centrifuge tube 21. Flush out remaining cells by washing column with an additional 2 ml of selection buffer 22. Remove LD column from magnet and discard 23. Spin down flow through at 3500 g at 4 C and aspirate supernatant 24. To resuspended yeast, add 5mL selection buffer containing fluorophore labeled antigen at a μm concentration (if using a secondary to stain preincubate the secondary with antigen before staining yeast and use a molar concentration of 2:3 secondary to antigen) 25. Rock or rotate slowly at 4 C for 1 hour 26. Spin down yeast for 3 minutes at 3500 g at 4 C and aspirate supernatant 27. Resuspend yeast in 4.5 ml of selection buffer and 500 μl of anti-fluorophore beads 28. Rock or rotate slowly at 4 C for 20 minutes 29. Approximately 5 minutes before end of incubation, place an LS column on the Miltenyi MACS magnet. Put a 15 ml sterile falcon tube under the column and place a 50mL cap over the top of the column to maintain sterility. Keep plunger wrapped in package for later use. Add 5 ml of selection buffer to the LS column and allow it to equilibrate before the next step. It will take ~5 minutes to flow through completely 30. Spin down yeast for 3 minutes at 3500 g at 4 C and aspirate buffer and unbound beads 31. Resuspend yeast by pipetting in 3 ml of selection buffer and spin down as above 32. Aspirate supernatant and resuspend yeast in 3 ml of selection buffer 33. Remove 50 μl pre-ls aliquot of resuspended yeast and set aside for later analysis. Keep aliquot on ice. 34. Flow yeast over the equilibrated LS column and collect flow through in a new sterile 15 ml centrifuge tube 35. Wash column with 8 ml of selection buffer

22 36. Record approx. volume of flow through and remove 50 μl flow through aliquot for later analysis. 37. Remove LS column from magnet and place over fresh sterile 15 ml centrifuge tube 38. Add 5 ml of selection buffer to column and immediately use plunger to elute cells 39. Remove 50 μl elution aliquot from eluted cells and set aside for later analysis. Keep aliquot on ice. 40. Spin down elution at 3500 g for 3 min at 4 C and aspirate buffer taking care not to disturb the pellet 41. Resuspend yeast in 3 ml of TRP +glucose in a 14 ml Falcon culture tube 42. Shake at 30 C for about 24 hours to recover. 43. Use flow cytometry to compare the number of yeast from pre-ls and elution aliquots and use this ratio to get an estimate of the reduction in diversity of the library. Next steps: After recovering yeast, you can reinduce in galactose medium for another round of selection, and iterate until you isolate binders. It s a good idea to use a different fluorophore for each selection round. We typically perform two MACS selections followed by a FACS sort to isolate binders. For a FACS sort, you can prepare your yeast by staining with antigen exactly as described above. The details of how to sort will depend on the specific project and sorting equipment being used. Once your selected library has a high proportion of active clones (usually 20% or more of the total cells), you can move on to screening. For screening, plate sorted yeast on TRP plates for single clones. Grow single clones in a sterile deep-well 96-well plate, with one clone per well. Then induce expression and stain clones to identify those with the highest activity. Finally, a yeast miniprep or colony PCR can be used to isolate nanobody DNA for analysis and recombinant expression. Note that not every clone with activity on yeast retains activity in recombinant form. A large majority of clones will show activity in both formats, but there are some exceptions.

23 Appendix Yglc4.5 Trp (1 liter) 3.8 g of Trp drop-out media supplement (US Biological) 6.7 g Yeast Nitrogen Base 10.4 g Sodium Citrate 7.4 g Citric Acid Monohydrate 10 ml Pen-Strep (10,000 units/ml stock) 20 g glucose Adjust ph to 4.5; Sterifilter (or sterifilter a stock of 20% glucose and add with antibiotic after autoclaving). Trp (+glucose or +galactose - 1 liter) 3.8 g Trp drop-out media supplement (US Biological) 6.7 g Yeast Nitrogen Base 10 ml Pen-Strep (10,000 units/ml stock) 20 g glucose or galactose (glucose for normal growth and galactose for induction of nanobodies) Adjust ph to 6; Sterifilter (or sterifilter a stock of 20% glucose or galactose and add with antibiotic after autoclaving). YPAD (1 liter) 20 g Bacto Peptone 20 g Glucose 10 g Yeast Extract 18 mg Adenine 10 ml Pen-Strep (10,000 units/ml stock) Selection buffer* 20 mm HEPES ph mm sodium chloride 0.1% (w/v) bovine serum albumin 5 mm maltose Sterile filter before use. *Many other buffers will work equally well, depending on your protein you may need to modify this. For GPCRs for instance, we usually add 0.1% (w/v) lauryl maltose neopentyl glycol detergent. No ph adjustment needed. Sterifilter or autoclave as appropriate.

24 MACS accessories catalog numbers Anti-Cy5/Anti-Alexa Fluor 647 MicroBeads Miltenyi Biotec Cat# Anti-FITC MicroBeads Miltenyi Biotec Cat# LD Columns Miltenyi Biotec Cat# LS Columns Miltenyi Biotec Cat#

25 Library vector sequence: Nb.BV025 in pyds649hm 8584 bp. Selectable in E. coli with ampicillin and in yeast by Trp dropout. Sequence with Gal1 promoter primer ( AATATACCTCTATACTTTAACGTC ). Mating factor alpha leader Nanobody coding sequence HA epitope tag 649 stalk sequence GGTACCCGACAGGTTATCAGCAACAACACAGTCATATCCATTCTCAATTAGCTCTACCA CAGTGTGTGAACCAATGTATCCAGCACCACCTGTAACCAAAACAATTTTAGAAGTACTT TCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTG GATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCC ATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTA TCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGC TATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGAC TCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGC GCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAG GAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAA CCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGA AGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTA ACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATC AACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGAT CGAATTCAACCCTCACTAAAGGGCGGCCGCCATGAGATTCCCATCTATCTTCACCGCTG TTTTGTTCGCTGCTTCTTCTGCTTTGGCTGCTCCAGCTAACACCACCACCGAAGACGAA ACCGCTCAAATCCCAGCTGAAGCTGTTATCGACTACTCTGACTTGGAAGGTGACTTCGA CGCTGCTGCTTTGCCATTGTCTAACTCTACCAACAACGGTTTGTCTTCTACCAACACCAC CATCGCTTCTATCGCTGCTAAGGAAGAAGGTGTTCAATTGGACAAGAGAGAAGCTAGC GCACAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTG CGCCTGAGCTGCGCGGCGAGCGGCTCTATTTCTCTGTACGCTGAAATGGGCTGGTATC GCCAGGCGCCGGGCAAAGAACGCGAACTTGTTGCCGGTATTACTCGTGGTACTACTAC CTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAAC ACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCG CGGCTGACTACGTTCAGGGTAAATCTCGTGTTATCCCGCATTACTATTGGGGCCAGGG CACCCAGGTGACCGTGAGCAGCGGATCCTACCCATACGATGTTCCAGATTACGCTCAA ACATCCTCACCCTCTACAGAAGCATCAGTATCAACCTCTAGTACCTCTTCCTCTGCTTCA CAATCATCTGACCCAACTACAACATCTTCGTCCAGTTCATCGTCTTCCCCATCTTCCCAA TCTGAGGAAATTTCATCATCACCGACGGTCTCAACTACACCATCAACTTCTTCATCATCT TCCTCAATGACTTCAACCACCACAACAAAGTCAATCTCAACTTCCACTACAAGTTCAGC TCCAGTTACAGATGTGACAGTTTCCTCATCGCCTAGTAAATCTACCTCTACTTCGACAA GTACAGAAACATCTAAAACACCTACTTCAATGACAGAGTATACATCTAGTACATCGATA ATTTCGACTCCAGTTAGTCACTCGCAGACAGGTTTGTCGGCTTCATCAAGTTCATCATC TACAACATCCGGTTCTTCGTCCACTAAATCAGAAAGTTCGACAACATCTGGCTCTTCCC AGTCCGTGGAATCAACCTCCAGCCACGCCACTGTTCTTGCTAATTCCGCAGAAATGGTC

26 ACAACATCCTCTAGTTCATCCTCAACATCCGAAATGTCATTAACTAGTACTGCTACCAG TGTACCAGTCTCATCTAGTAGCAGTACGACATATTCTACTAGCGCATCTACACAAGCCG TCACTACAACATCTTCTTCCACTGTATCTACAACTTCTTCTAGTACAACGTTAACAAGCG CATTCACACATTCTTCAACCACATCGTCCGACCAGCCACCCAGCGACACTACAAGTCCA TCTACGACACACGAACCTCATGTAACCACTCAGACGTCATCAGAAACATCTTCTTCTAA GTCATCTTCTACTTCTTCTTCAAGTACATCTCAAACCTCTGAGTCTGCAACACCATCCGA TTCCGTATCACCTGGAAGTTCTACATCAACATCTTCTAGTAGCACTTCTACTTCCACTTC TATTTCCAGTGGAGAAACGACAACTTCTTCTTCTTCATCATCTGCCACGACCACTTCTAA CAGCGCAACCTTGTCAGTCTCTACCACACAAACTTCGATTGAAGCCAGTTCATCTACTA CATCTACATCTAGTTCAACAATTACAACTTCAAGTAGTAGCGCTCACATATCGTCGAAA TCTCAATCTAGTATTACCTATCCCTCTTCCTCGACATCTTCATCTACATCGTCCTCAATTT CTAGCGAATCTGAAAGTTTTGAATCGACATCAGCAGAAGATGCTCCATCAACAGCACC TTCATCAAGTGTCTCTTCTAAGAGTTCTACCTCTACAACATCAAGCACATCGACATCTTC AAGCACTCCATCTCCATCACCATCTTCCGTGAGTTCTTCCTCCACCAGCTCATTGACAAC TTCTGCTGTATCAACACCAGCTACCTCTCATTCTCAAAGTACTGTAGTAACCACCACTA CTATTACTACATCAACAGGTCCAGTGATGTCTACGACAACAGCTTATTCTTCTAGTTCT ACTAGCAGCTCGGAATCTTCTGAGGTTCAGTCTGTCATGTCATCTACGCCTAGTTCAAC ATCAACAACAACCAGTTCGGAATCTACTTCATCTAGCTCCACAGCTTCTACCTCACCAT CAACCTCGCAAACTTTCGAAACTTCTCCTACTATAGGAGGTGTCCCCTCAACCACTTCA TTTGTCTCTACGCCAACAACGAAATTGTCGCACACTACTTCCACTATGACAGCACAGTC CGATAGTAAGTCTACCCACTCCTCAAGCACATCGACAGAAGATAAATCATCCACTGCTT CTGCAGTTGACGAAAGCACTACAACATCCACTTCCACGGAGTCTACTACATCAGTAACA TCAGGCACCTCCCATTCCGCTAAAGAATCTTCGTCAAATTCTAAGGTGTATAGTTCACA GACAGCACACTCATCCATAAGTGTTGCATCATCACCTAGTACAAAGGGCGCCCAAATC CAATCTTCTATGGTTGAAATCTCTACCTACGCTGGTTCTGCTAACTCTGTTAACGCTGG TGCTGGTGCTGGTGCTTTGTTCTTGTTGTTGTCTTTGGCTATCATCTAATGATTAATTAA CTCGAGATCTGATAACAACAGTGTAGATGTAACAAAATCGACTTTGTTCCCACTGTACT TTTAGCTCGTACAAAATACAATATACTTTTCATTTCTCCGTAAACAACATGTTTTCCCAT GTAATATCCTTTTCTATTTTTCGTTCCGTTACCAACTTTACACATACTTTATATAGCTATT CACTTCTATACACTAAAAAACTAAGACAATTTTAATTTTGCTGCCTGCCATATTTCAATT TGTTATAAATTCCTATAATTTATCCTATTAGTAGCTAAAAAAAGATGAATGTGAATCGA ATCCTAAGAGAATTGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGC CGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTG CAGCACATCCCCCCTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCC TTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCGCA TTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCC TAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTC GACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGA CGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAA ACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCC GATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTA ACAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCG GTATTTCACACCGCAGGCAAGTGCACAAACAATACTTAAATAAATACTACTCAGTAATA

27 ACCTATTTCTTAGCATTTTTGACGAAATTTGCTATTTTGTTAGAGTCTTTTACACCATTT GTCTCCACACCTCCGCTTACATCAACACCAATAACGCCATTTAATCTAAGCGCATCACC AACATTTTCTGGCGTCAGTCCACCAGCTAACATAAAATGTAAGCTTTCGGGGCTCTCTT GCCTTCCAACCCAGTCAGAAATCGAGTTCCAATCCAAAAGTTCACCTGTCCCACCTGCT TCTGAATCAAACAAGGGAATAAACGAATGAGGTTTCTGTGAAGCTGCACTGAGTAGTA TGTTGCAGTCTTTTGGAAATACGAGTCTTTTAATAACTGGCAAACCGAGGAACTCTTGG TATTCTTGCCACGACTCATCTCCATGCAGTTGGACGATATCAATGCCGTAATCATTGAC CAGAGCCAAAACATCCTCCTTAGGTTGATTACGAAACACGCCAACCAAGTATTTCGGA GTGCCTGAACTATTTTTATATGCTTTTACAAGACTTGAAATTTTCCTTGCAATAACCGG GTCAATTGTTCTCTTTCTATTGGGCACACATATAATACCCAGCAAGTCAGCATCGGAAT CTAGAGCACATTCTGCGGCCTCTGTGCTCTGCAAGCCGCAAACTTTCACCAATGGACCA GAACTACCTGTGAAATTAATAACAGACATACTCCAAGCTGCCTTTGTGTGCTTAATCAC GTATACTCACGTGCTCAATAGTCACCAATGCCCTCCCTCTTGGCCCTCTCCTTTTCTTTT TTCGACCGAATTAATTCTTAATCGGCAAAAAAAGAAAAGCTCCGGATCAAGATTGTAC GTAAGGTGACAAGCTATTTTTCAATAAAGAATATCTTCCACTACTGCCATCTGGCGTCA TAACTGCAAAGTACACATATATTACGATGCTGTCTATTAAATGCTTCCTATATTATATA TATAGTAATGTCGTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTA AGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCC CGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTT TTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTA TAGGTTAATGTCATGATAATAATGGTTTCTTAGGACGGATCGCTTGCCTGTAACTTACA CGCGCCTCGTATCTTTTAATGATGGAATAATTTGGGAATTTACTCTGTGTTTATTTATTT TTATGTTTTGTATTTGGATTTTAGAAAGTAAATAAAGAAGGTAGAAGAGTTACGGAAT GAAGAAAAAAAAATAAACAAAGGTTTAAAAAATTTCAACAAAAAGCGTACTTTACATA TATATTTATTAGACAAGAAAAGCAGATTAAATAGATATACATTCGATTAACGATAAGT AAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCTACACAGACAAGATGAAACAA TTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAGTATTTGTTGGCGA TCCCCCTAGAGTCTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTTTT TTTACTTTCTATTTTTAATTTATATATTTATATTAAAAAATTTAAATTATAATTATTTTTA TAGCACGTGATGAAAAGGACCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCC CTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCT GATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGT CGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCT GGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACT GGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCA AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCA TAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTTCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGC AATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGC

28 GGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA CGACGGGCAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGC ACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC ATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGGAACGCCTGGTATC TTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCG TCAGGGGGGCCGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCG GGCAGTGAGCGCAACGCAATTAATGTGAGTTACCTCACTCATTAGGCACCCCAGGCTT TACACTTTATGCTTCCGGCTCCTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC ACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGGAATTAACCCTCACTAAAGGG AACAAAAGCTG