title: Optimization of the crystallizability of a single-chain antibody fragment.
abstract: Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.
authors.affiliation: Institute of Molecular Genetics, ASCR, v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Škerlová
authors.affiliation: Institute of Molecular Genetics, ASCR, v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Král
authors.affiliation: Institute of Molecular Genetics, ASCR, v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Milan
authors.initials: M
authors.lastname: Fábry
authors.affiliation: Institute of Molecular Genetics, ASCR, v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Juraj
authors.initials: J
authors.lastname: Sedláček
authors.affiliation: Institute of Organic Chemistry and Biochemistry, ASCR, v.v.i., Flemingovo nám. 2, 16610 Prague 6, Czech Republic.
authors.forename: Václav
authors.initials: V
authors.lastname: Veverka
authors.affiliation: Institute of Molecular Genetics, ASCR, v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Pavlína
authors.initials: P
authors.lastname: Rezáčová
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003460:Crystallization; D006801:Humans; D008969:Molecular Sequence Data; D057127:Single-Chain Antibodies / Q000737:chemistry*
chemical_list: D057127:Single-Chain Antibodies
keywords: Thermofluor assay; crystallizability optimization; crystallization; differential scanning fluorimetry; oligomerization; single-chain antibody fragment