title: Assembly of a functional immunoglobulin Fv fragment in Escherichia coli.
abstract: An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli. The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the Fv fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The Fv fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant Fv fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the Fv fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.
authors.affiliation: Genzentrum der Universität München, Max-Planck-Institut für Biochemie, Martinsried, FRG.
authors.forename: A
authors.initials: A
authors.lastname: Skerra
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000302:isolation & purification; D009194:Myeloma Proteins / Q000235:genetics / Q000276:immunology; D010767:Phosphorylcholine; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D009194:Myeloma Proteins; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D010767:Phosphorylcholine