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A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries

  • Davide FerrariEmail author
  • Valentina Garrapa
  • Massimo Locatelli
  • Angelo Bolchi
Original paper

Abstract

Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time- and resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in E. coli when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 1012 that is three orders of magnitude higher than common phage display libraries. The bacterial expression of several library clones showed a high production of soluble recombinant proteins. The high complexity of the library, confirmed by sequencing of a subset of clones, as well as a preliminary RD selection of a maltose binding protein binder, indicated this approach as a starting point in the construction of synthetic combinatorial libraries to be used as animal-free tools for the low-cost selection of target-specific nanobodies.

Keywords

Nanobody VHH Scaffold Library Ribosome display 

Notes

Acknowledgement

V.G. is the recipient of a PhD student fellowship from the Fondazione Cariparma. This work was supported in part by a grant from Regione Emilia-Romagna, Italy (Programma di Ricerca Regione-Università 2010-2012; PRUa1RI-2012-006). Support from the Interuniversity Consortium for Biotechnologies (CIB) and European Molecular Biology Organization (EMBO) is also gratefully acknowledged.

Funding

This work has benefited from the equipment and framework of the COMP-HUB Initiative, funded by the ‘Departments of Excellence’ program of the Italian Ministry for Education, University, and Research (MIUR, 2018-2022).

Compliance with Ethical Standards

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Supplementary material

12033_2019_224_MOESM1_ESM.docx (1.3 mb)
Supplementary material 1 (DOCX 1315 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry, Life Sciences and Environmental SustainabilityUniversity of ParmaParmaItaly
  2. 2.San Raffaele HospitalMilanItaly
  3. 3.Biopharmanet-Tec LaboratoryUniversity of ParmaParmaItaly

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