SEQUENCE-ANALYSIS AND BACTERIAL PRODUCTION OF THE ANTI-C-MYC ANTIBODY9E10 - THE V-H DOMAIN HAS AN EXTENDED CDR-H3 AND EXHIBITS UNUSUAL SOLUBILITY

The cDNAs for the two variable domains of the antibody 9E10 were clone d from the hybridoma cell line. A chimeric 9E10 F-ab fragment was prod uced in E. coli under control of the tightly controlled tetracycline p romoter. The functional F-ab fragment was isolated in a single step vi a a His(6)-tag, which also served for its recognition by a nickel chel ate-alkaline phosphatase conjugate. Thus, the recombinant F-ab fragmen t permitted the immunochemical detection of the myc tag in a sandwich ELISA, The dissociation constant for the interaction with the myc tag peptide was determined as 80 +/- 5 nM by fluorescence titration, In an attempt to produce the smaller 9E10 F-v fragment it was found that it s V-H domain alone can be readily isolated from E. coli as a soluble p rotein. This unusual behaviour may be explained by the 18 amino acid-l ong CDR-H3 and could be of value in the design of 'single domain' anti bodies. (C) 1997 Federation of European Biochemical Societies.