Engineering stable cytoplasmic intrabodies with designed specificity.
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.
Donini
Marcello
M
ENEA, UTS Biotecnologie, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, P.O. Box 2400 I-00100, Roma, Italy.
Morea
Veronica
V
Desiderio
Angiola
A
Pashkoulov
Dimitre
D
Villani
Maria Elena
ME
Tramontano
Anna
A
Benvenuto
Eugenio
E
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
D000939:Epitopes; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D009113:Muramidase