A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold

TitleA semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold
Publication TypeArticolo su Rivista peer-reviewed
Year of Publication2001
AuthorsDesiderio, Angiola, Franconi Rosella, Lopez M., Villani Maria Elena, Viti F., Chiaraluce R., Consalvi V., Neri D., and Benvenuto Eugenio
JournalJournal of Molecular Biology
Volume310
Pagination603-615
ISSN00222836
KeywordsAmino Acid Sequence, Antibody, antibody production, antigen binding, article, Bacteria (microorganisms), binding affinity, binding site, Escherichia coli, immunochemistry, molecular cloning, nonhuman, Phage display, priority journal, protein stability, synthesis
Abstract

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 5 × 107 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. © 2001 Academic Press.

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URLhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-0035854543&doi=10.1006%2fjmbi.2001.4756&partnerID=40&md5=7f6e892ed945d203373ba9994ad4ce16
DOI10.1006/jmbi.2001.4756