Generation of synthetic nanobodies against delicate proteins


Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10–30 unique sybodies exhibiting binding affinities in the range of 500 pM–500 nM.

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Fig. 1: Sybody libraries.
Fig. 2: Sybody selection flowchart.
Fig. 3: Overview of genetic constructs and primers.
Fig. 4: Exemplary DNA gel of sybody pools.
Fig. 5: SEC analysis of sybodies.
Fig. 6: A conformation-specific sybody against the ABC transporter TM287/288.
Fig. 7: Structure of the KDEL receptor in complex with a sybody.

Data availability

All plasmids have been deposited on Addgene. The sybody libraries can be obtained from the authors via an academic material transfer agreement.


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We wish to thank all members of the Seeger and Geertsma laboratoriess for scientific discussions. We acknowledge Jenifer Cuesta Bernal for critical reading. E.R.G. acknowledges financial support from the German Research Foundation via the Cluster of Excellence Frankfurt (Macromolecular Complexes) and the CRC807 (Transport and Communication across Biological Membranes). Work in the Seeger group was supported by an SNSF Professorship of the Swiss National Science Foundation (PP00P3_144823, to M.A.S.), an SNSF NRP 72 grant (407240_177368, to M.A.S.), an SNSF BRIDGE proof-of-concept grant (20B1-1_175192, to P.E.) and a BioEntrepreneur-Fellowship of the University of Zurich (BIOEF-17-002, to I.Z.). R.J.P.D, E.R.G., and M.A.S. acknowledge a grant from the Commission for Technology and Innovation CTI (16003.1 PFLS-LS). Work in the group of S.N. was supported by a Wellcome award (102890/Z/13/Z).

Author information




M.A.S., E.R.G. and R.J.P.D. conceived the sybody project. E.R.G. and M.A.S. designed the sybody library. I.Z. and P.E. established the sybody selection platform. C.A.J.H. established the ELISA setup. I.Z., C.A.J.H., B.T.K., P.B. and E.R.G. selected sybodies against protein targets. I.Z., S.N., E.R.G. and M.A.S. supervised students and postdocs. I.Z., B.T.K., E.R.G. and M.A.S. wrote the manuscript. P.E., C.A.J.H., S.N. and R.J.P.D. edited the manuscript.

Corresponding authors

Correspondence to Eric R. Geertsma or Markus A. Seeger.

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Competing interests

The authors declare competing financial interests. I.Z., P.E., R.J.P.D. and M.A.S. are co-founders and shareholders of Linkster Therapeutics AG.

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Peer review information Nature Protocols thanks Serge Muyldermans, Jamshid Tanha and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Zimmermann, I. et al. Elife 7, e34317 (2018):

Hutter, C. et al. Nat. Commun. 10, 2260 (2019):

Bräuer, P. et al. Science 363, 1103–1107 (2019):

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Zimmermann, I., Egloff, P., Hutter, C.A.J. et al. Generation of synthetic nanobodies against delicate proteins. Nat Protoc 15, 1707–1741 (2020).

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