Bacterial expression of immunoglobulin VH proteins.
A bacterial expression system in Escherichia coli has been developed that produces as much as 10 mg/l of culture of the VH protein associated with monoclonal antibodies specific for the 5-dimethylaminonaphthalene-2-sulfonyl (Dns) group. This system has been applied to the expression of the VH genes derived from a low-affinity, IgM-producing hybridoma and from a high-affinity, IgG-producing cell line. The plasmid vectors (contributed by Dr William F. Studier) utilize a T7 expression cassette whose activity is initiated by infection with a lambda phage derivative carrying the T7 RNA polymerase gene. The VH proteins were extracted from the bacterial pellet in 8 M urea and purified by chromatography in 8 M urea. Recombinants with the homologous light (L) chains were prepared to yield VHL molecules. These were used to measure intrinsic affinity for Dns-lysine by resonance energy transfer. The association constants were 7 x 10(6) M-1 and 7 x 10(9) M-1 for the low- and high-affinity systems, respectively. These values are not significantly different from those observed with monoclonal antibodies secreted from the corresponding cell lines. This system lends itself to the quantitative evaluation of the binding properties of the VH protein itself as well as the modulation of affinity by site-directed mutagenesis.
Udaka
K
K
Department of Microbiology, School of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Chua
M M
MM
Tong
L H
LH
Karush
F
F
Goodgal
S H
SH
D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D005798:Genes, Bacterial; D007143:Immunoglobulin Heavy Chains / Q000032:analysis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000032:analysis* / Q000235:genetics
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region