Antibodies specific for (6-4) DNA photoproducts: cloning, antibody modeling and construction of a single-chain Fv derivative.
We have investigated a series of four monoclonal antibodies that specifically recognize pyrimidine (6-4) pyrimidone photoproducts. One of these antibodies (64M4), bound all four possible pyrimidine-pyrimidone photoadducts with equal affinities whereas the others (64M2, 64M3 and 64M5) were selective for TC and TT sequences. In addition, 64M5 had the highest binding affinity for photodamaged DNA of the four [T. Mori et al., Photochem. Photobiol. 54 (1991) 225-232]. To help understand the differences between these antibodies, we have cloned and sequenced the variable region genes from all four. Comparing these sequences revealed that all four were highly similar to one another, although there were some differences in potential antigen-contact regions. To assess the influences of these sequence differences at the structural level, computer models were constructed for all four antibodies. Most of the sequence differences occurred in potential antigen contact regions, suggesting specific positions that might account for the observed differences in binding affinities and selectivities. A single-chain Fv derivative of 64M5 was therefore constructed and characterized to provide an experimental system in which structure-function relationships can be tested. This derivative could be isolated from Escherichia coli using two chromatographic steps and possessed the same binding specificity as the parent monoclonal antibody.
Morioka
H
H
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan.
Miura
H
H
Kobayashi
H
H
Koizumi
T
T
Fujii
K
K
Asano
K
K
Matsunaga
T
T
Nikaido
O
O
Stewart
J D
JD
Ohtsuka
E
E
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D004247:DNA / Q000528:radiation effects*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000528:radiation effects; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011740:Pyrimidine Dimers / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014466:Ultraviolet Rays
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009841:Oligonucleotides; D011740:Pyrimidine Dimers; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; C058406:pyrimidine-pyrimidone dimer; D004247:DNA