|Title||A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies.|
|Publication Type||Journal Article|
|Year of Publication||2012|
|Authors||Pellis, M., E. Pardon, K. Zolghadr, U. Rothbauer, C. Vincke, J. Kinne, I. Dierynck, K. Hertogs, H. Leonhardt, J. Messens, S. Muyldermans, and K. Conrath|
|Journal||Arch Biochem Biophys|
|Date Published||2012 Oct 15|
|Keywords||Amino Acid Sequence, Animals, Antibody Affinity, Camels, Cell Line, Cloning, Molecular, Escherichia coli, Gene Expression, Green Fluorescent Proteins, HIV Integrase, HIV-1, Immunoglobulin Fragments, Molecular Sequence Data, N-Glycosyl Hydrolases, Peptide Library, Protein Stability, Trypanosoma vivax|
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.
|Alternate Journal||Arch. Biochem. Biophys.|
A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies.