Expression, purification and characterization of B72.3 Fv fragments.
The Fv fragment of the antibody B72.3 has been produced by expression in both a mammalian and microbial system, namely Chinese hamster ovary (CHO) cells and Escherichia coli. In both cases secretion of the Fv into the culture medium was achieved, with equivalent amounts of Vh and Vl produced. The yield of Fv from CHO cells was 4 mg/l in roller-bottle culture. E. coli proved to be a more productive system with yields of 40 mg/l in shake flasks rising to 450 mg/l in fermentations. B72.3 Fv from both sources was capable of binding to antigen with similar binding ability to the Fab' fragment. A detailed sedimentation analysis, both by velocity and equilibrium techniques, revealed that the two domains of Fv are associated at high concentrations at pH values close to neutral, but dissociate at concentrations lower than approx. 0.5 mg/ml. Individual Vh or Vl polypeptides are not able to bind to the antigen and thus these results suggest that the antigen promotes assembly of Fv at the low concentrations used in the antigen-binding assays. At a pH value of 1.9, Vh and Vl are completely dissociated even at very high concentrations and are apparently unfolded at low solute concentrations. Small-angle X-ray scattering was used to measure a radius of gyration of 1.75 +/- 0.2 nm (17.5 +/- 2 A) for Fv.
King
D J
DJ
Celltech Ltd., Slouth, Berks, U.K.
Byron
O D
OD
Mountain
A
A
Weir
N
N
Harvey
A
A
Lawson
A D
AD
Proudfoot
K A
KA
Baldock
D
D
Harding
S E
SE
Yarranton
G T
GT
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000302:isolation & purification; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014461:Ultracentrifugation
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins