title: Crystallization of single-chain Fv proteins.
abstract: Single-chain Fv (sFv) proteins consist of the variable heavy chain (VH) and variable light chain (VL) domains of an antibody, covalently joined by an engineered polypeptide linker. We report the crystallization of single-chain Fv's with specificities for fluorescein (4-4-20 sFv) and the TAG-72 pan-carcinoma glycoprotein antigen (CC49 sFv). Concentration of these proteins, preliminary to crystallization, results in a monomer-multimer equilibrium, causing aggregation which interferes with crystallization. Aggregation has been observed to depend primarily on an intact linker between VL and VH domains, although other factors are likely to modulate this phenomenon as well, including the specific identity of Fv and ligand, presence or absence of the ligand, linker length and possibly sequence. We have found two methods to overcome sFv aggregation, both of which yield X-ray diffraction quality crystals. The first, discovered serendipitously, is by introducing a proteolytic clip into the linker region (effectively yielding an Fv fragment). The second is the purification of the sFv dimer form, with linker regions intact, from an equilibrium mixture of aggregates. The sFv molecular association in a dimer is believed to be unusual in that each VL/VH interface may not be formed by the two linker-connected VL and VH domains, but rather by interaction of VL and VH domains from two distinct sFv monomers. Structure determination of the CC49 sFv dimer, with the 14-residue linker designated 212, is underway to test this model. Increasing linker length, to relieve steric strain on the monomer, and inclusion of the appropriate antigen, to slow transitions between monomeric and multimeric forms, may prove valuable strategies with sFv proteins less amenable to crystallization.
authors.affiliation: Protein Engineering Department Enzon Incorporated, Piscataway, NJ 08854-3998.
authors.forename: N Z
authors.initials: NZ
authors.lastname: Essig
authors.forename: J F
authors.initials: JF
authors.lastname: Wood
authors.forename: A J
authors.initials: AJ
authors.lastname: Howard
authors.forename: R
authors.initials: R
authors.lastname: Raag
authors.forename: M
authors.initials: M
authors.lastname: Whitlow
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000936:Antigen-Antibody Complex; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D018360:Crystallography, X-Ray; D019793:Fluorescein; D005452:Fluoresceins; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000302:isolation & purification; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000951:Antigens, Neoplasm; D005452:Fluoresceins; D006023:Glycoproteins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; C047052:tumor-associated antigen 72; D019793:Fluorescein