title: Engineering stable cytoplasmic intrabodies with designed specificity.
abstract: Many attempts have been made to develop antibody fragments that can be expressed in the cytoplasm ("intrabodies") in a stable and functional form. The recombinant antibody fragment scFv(F8) is characterised by peculiarly high in vitro stability and functional folding in both prokaryotic and eukaryotic cytoplasm. To dissect the relative contribution of different scFv(F8) regions to cytoplasmic stability and specificity we designed and constructed five chimeric molecules (scFv-P1 to P5) in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffold. All five chimeric scFvs were expressed in a soluble form in the periplasm and cytoplasm of Escherichia coli. All the periplasmic oxidised forms and the scFv(P3) extracted from the cytoplasm in reducing conditions had HEL binding affinities essentially identical (K(d)=15nM) to that of the cognate scFv(D1.3) fragment (K(d)=16nM). The successful grafting of the antigen binding properties of D1.3 onto the scFv(F8) opens the road to the exploitation of this molecule as a scaffold for the reshaping of intrabodies with desired specificities to be targeted to the cytoplasm.
authors.affiliation: ENEA, UTS Biotecnologie, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, P.O. Box 2400 I-00100, Roma, Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Donini
authors.forename: Veronica
authors.initials: V
authors.lastname: Morea
authors.forename: Angiola
authors.initials: A
authors.lastname: Desiderio
authors.forename: Dimitre
authors.initials: D
authors.lastname: Pashkoulov
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Villani
authors.forename: Anna
authors.initials: A
authors.lastname: Tramontano
authors.forename: Eugenio
authors.initials: E
authors.lastname: Benvenuto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001667:Binding, Competitive; D002645:Chickens; D003593:Cytoplasm / Q000276:immunology*; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology; D016296:Mutagenesis; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000939:Epitopes; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D009113:Muramidase