title: Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display.
abstract: Intracellular antibodies (intrabodies) provide an attractive means for manipulating intracellular protein function, both for research and potentially for therapy. A challenge in the isolation of effective intrabodies is the ability to find molecules that exhibit sufficient binding affinity and stability when expressed in the reducing environment of the cytoplasm. Here, we have used yeast surface display of proteins to isolate novel scFv clones against huntingtin from a non-immune human antibody library. We then applied yeast surface display to affinity mature this scFv pool and analyze the location of the binding site of the mutant with the highest affinity. Interestingly, the paratope was mapped exclusively to the variable light chain domain of the scFv. A single domain antibody was constructed consisting solely of this variable light chain domain, and was found to retain full binding activity to huntingtin. Cytoplasmic expression levels in yeast of the single domain were at least fivefold higher than the scFv. The ability of the single-domain intrabody to inhibit huntingtin aggregation, which has been implicated in the pathogenesis of Huntington's disease (HD), was confirmed in a cell-free in vitro assay as well as in a mammalian cell culture model of HD. Significantly, a single-domain intrabody that is functionally expressable in the cytoplasm was derived from a non-functional scFv by performing affinity maturation and binding site analysis on the yeast cell surface, despite the differences between the cytoplasmic and extracellular environment. This approach may find application in the development of intrabodies to a wide variety of intracellular targets.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Colby
authors.forename: Payal
authors.initials: P
authors.lastname: Garg
authors.forename: Tina
authors.initials: T
authors.lastname: Holden
authors.forename: Ginger
authors.initials: G
authors.lastname: Chao
authors.forename: Jack M
authors.initials: JM
authors.lastname: Webster
authors.forename: Anne
authors.initials: A
authors.lastname: Messer
authors.forename: Vernon M
authors.initials: VM
authors.lastname: Ingram
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: D000595:Amino Acid Sequence; D022801:Complementarity Determining Regions; D006801:Humans; D000071058:Huntingtin Protein; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics*; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D022801:Complementarity Determining Regions; C086055:HTT protein, human; D000071058:Huntingtin Protein; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins