Expression, characterization and crystallization of the Fv fragment of mouse antibody 3B62 from the secondary immune response.

Affinity of antibodies increases in the course of the immune response. Mouse anti-nitrophenol antibody 3B62 from the secondary immune response shows higher affinity than the primary-response antibodies. An expression system for the 3B62 Fv fragment was constructed by introducing coding regions for the V(L) and V(H) into the genome of the methylotrophic yeast Pichia pastoris. Each of the coding regions was placed downstream of the coding region for the secretion signal of the yeast alpha-factor. The alpha-factor signals were cleaved off from the expressed proteins and the Fv was secreted as a heterodimer consisting of the V(L) and V(H) domains. The binding constant of the expressed Fv against the (4-hydroxy-5-iodo-3-nitrophenyl)acetate ligand was comparable to that of the Fab fragment. Crystals of the Fv were obtained in the presence of the ligand and diffracted X-rays to 1.8 A resolution. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 46.48 (9), b = 34.99 (4), c = 77.76 (17) A, beta = 101.47 (14) degrees, and contain one Fv molecule per asymmetric unit.