A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies

Archives of Biochemistry and Biophysics
Mireille PellisKatja Conrath

Abstract

Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30 kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.

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Citations

Jun 5, 2013·Nanomedicine·Gholamreza Hassanzadeh-GhassabehSerge Muyldermans
Apr 5, 2014·Trends in Biotechnology·Thomas De MeyerAnn Depicker
Mar 19, 2013·Annual Review of Biochemistry·Serge Muyldermans
Oct 16, 2012·Cellular and Molecular Life Sciences : CMLS·Filippa FleetwoodJohn Löfblom
Nov 2, 2013·Proteomics. Clinical Applications·Michael J TaussigOda Stoevesandt
Dec 10, 2015·Biotechnology Journal·Songhee LeeMasahiro Kawahara
Jan 21, 2014·Protein Expression and Purification·Ellen R GoldmanGeorge P Anderson
Mar 19, 2014·Journal of Structural Biology·Tim KükenshönerKatja M Arndt
Sep 5, 2012·Archives of Biochemistry and Biophysics·Florian Rüker
Sep 8, 2016·PloS One·Lucía MaffeyViviana Parreño
Mar 9, 2017·Protein Science : a Publication of the Protein Society·Thomas Böldicke
Sep 20, 2018·Molecular & Cellular Proteomics : MCP·Bettina-Maria KellerBjoern Traenkle
Jun 8, 2019·SLAS Discovery·James Woods
Aug 12, 2020·The FEBS Journal·Serge Muyldermans
Aug 3, 2014·Molecular Biology Reports·Aya TwairAbdul Qader Abbady
Dec 14, 2016·Annual Review of Pharmacology and Toxicology·Aashish ManglikJan Steyaert
Apr 18, 2020·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Congcong ZhangAndrea L J Marschall
Sep 9, 2017·Frontiers in Immunology·Bjoern Traenkle, Ulrich Rothbauer
Nov 26, 2020·Annual Review of Animal Biosciences·Serge Muyldermans
Nov 27, 2020·Frontiers in Cellular Neuroscience·Marit A de Beer, Ben N G Giepmans
Dec 17, 2020·Biomolecules·Eline SoetensXavier Saelens

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