A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold.
We report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. Four amino acid residues of the third hypervariable loop (CDR3) of both VH and VL were combinatorially mutated, generating a repertoire of approximately 5x10(7) independent scFvs, cloned in a phagemid vector. The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens. Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target. High yields of both soluble and phage antibodies were obtained in Escherichia coli. Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm. The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications.
Desiderio
A
A
Divisione Biotecnologie e Agricoltura, ENEA, C.R. Casaccia, Roma, I-00100, Italy.
Franconi
R
R
Lopez
M
M
Villani
M E
ME
Viti
F
F
Chiaraluce
R
R
Consalvi
V
V
Neri
D
D
Benvenuto
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000916:Antibody Diversity / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D000941:Antigens / Q000276:immunology; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003429:Cross Reactions / Q000276:immunology; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010084:Oxidation-Reduction; D019151:Peptide Library; D011489:Protein Denaturation / Q000187:drug effects; D015202:Protein Engineering; D020673:Protein Renaturation; D012995:Solubility; D013816:Thermodynamics
D000941:Antigens; D022801:Complementarity Determining Regions; D004220:Disulfides; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D019791:Guanidine