Epitope mapping of rat cytochrome P450 2B1 using glutathione S-transferase-2B1 fusion constructs.
We have previously produced 12 monoclonal antibodies (MAbs) against rat cytochrome P450(CYP)2B1, and 8 of these inhibit CYP2B1 catalytic activity to varying extents. Using competitive binding studies we showed that this collection of 12 MAbs recognize at least 6 spatially distinct epitopes. The complete coding sequence of CYP2B1 DNA in plasmid pSR-P450 was inserted into a glutathione S-transferase (GST) expression vector pGEX-1lambdaT so that it was in frame with the GST gene. Expression of GST-CYP2B1 was detected with most of the MAbs in Western blots except those which were conformation-specific. Fourteen different constructs were then made using PCR with oligonucleotide primers having EcoRI sites at their ends and were introduced into the GST expression vector at the EcoRI site. Each fusion construct was expressed in Escherichia coli, subjected to SDS-PAGE, blotted, and probed individually with each MAb. MAbs, which inhibited catalytic activity and were mutually competitive in binding to CYP2B1 (viz. BEA33, BE44, BE45, and BE28), recognized several fusion constructs and by deduction recognized amino acids 250-261 in CYP2B1. Other antibodies inhibiting catalytic activity recognized amino acids 262-272 (BEF29) and 306-491 (BE46, B50, and BE49) on CYP2B1. Non-inhibitory MAbs BE26 and BE32 were mapped to region 380-398 in CYP2B1. It was interesting to note that MAbs BEA33 and BE26, which also recognize spatially distinct epitopes on human CYP2E1 but not rat CYP2E1, had corresponding regions of high homology in human CYP2E1 but not rat CYP2E1. Identifying the epitopes recognized by this collection of MAbs will add to our understanding the sequences that may be important for producing inhibitory and specific antibodies to closely related antigens.
Parimoo
B
B
Chemical Biology, College of Pharmacy, Laboratory for Cancer Research, Rutgers The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, New Jersey 08854-8020, USA.
Thomas
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D019362:Cytochrome P-450 CYP2B1 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D019362:Cytochrome P-450 CYP2B1; D005982:Glutathione Transferase