title: A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies.
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.
authors.affiliation: Laboratory Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium.
authors.forename: Mireille
authors.initials: M
authors.lastname: Pellis
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.forename: Kourosh
authors.initials: K
authors.lastname: Zolghadr
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
authors.forename: Cécile
authors.initials: C
authors.lastname: Vincke
authors.forename: Joerg
authors.initials: J
authors.lastname: Kinne
authors.forename: Inge
authors.initials: I
authors.lastname: Dierynck
authors.forename: Kurt
authors.initials: K
authors.lastname: Hertogs
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.forename: Joris
authors.initials: J
authors.lastname: Messens
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Katja
authors.initials: K
authors.lastname: Conrath
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D002162:Camelus / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D019427:HIV Integrase / Q000276:immunology; D015497:HIV-1 / Q000201:enzymology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000276:immunology; D019151:Peptide Library; D055550:Protein Stability; D016689:Trypanosoma vivax / Q000201:enzymology
chemical_list: D007128:Immunoglobulin Fragments; D019151:Peptide Library; D049452:Green Fluorescent Proteins; D019427:HIV Integrase; D009699:N-Glycosyl Hydrolases