ANTIBODIES SPECIFIC FOR (6-4) DNA PHOTOPRODUCTS - CLONING, ANTIBODY MODELING AND CONSTRUCTION OF A SINGLE-CHAIN FV DERIVATIVE

Citation
H. Morioka et al., ANTIBODIES SPECIFIC FOR (6-4) DNA PHOTOPRODUCTS - CLONING, ANTIBODY MODELING AND CONSTRUCTION OF A SINGLE-CHAIN FV DERIVATIVE, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1385(1), 1998, pp. 17-32
Citations number
69
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,Biophysics
ISSN journal
0167-4838
Volume
1385
Issue
1
Year of publication
1998
Pages
17 - 32
Database
ISI
SICI code
0167-4838(1998)1385:1<17:ASF(DP>2.0.ZU;2-S
Abstract
We have investigated a series of four monoclonal antibodies that speci fically recognize pyrimidine (6-4) pyrimidone photoproducts. One of th ese antibodies (64M4), bound all four possible pyrimidine-pyrimidone p hotoadducts with equal affinities whereas the others (64M2, 64M3 and 6 4M5) 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 th e variable region genes from all four. Comparing these sequences revea led that all four were highly similar to one another, although there w ere some differences in potential antigen-contact regions. To assess t he influences of these sequence differences at the structural level, c omputer models were constructed for all four antibodies. Most of the s equence differences occurred in potential antigen contact regions, sug gesting specific positions that might account for the observed differe nces in binding affinities and selectivities. A single-chain Fv deriva tive 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 a s the parent monoclonal antibody. (C) 1998 Elsevier Science B.V. All r ights reserved.