A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies |
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Authors: | Pellis Mireille Pardon Els Zolghadr Kourosh Rothbauer Ulrich Vincke Cécile Kinne Joerg Dierynck Inge Hertogs Kurt Leonhardt Heinrich Messens Joris Muyldermans Serge Conrath Katja |
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Institution: | Laboratory Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium; VIB, Department of Structural Biology, Brussels, Belgium. |
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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 30kJ/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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