Sequence analysis and bacterial production of the anti-c-myc antibody 9E10: 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 cloned from the hybridoma cell line. A chimeric 9E10 F-ab fragment was produced in E. coli under control of the tightly controlled tetracycline promoter. The functional F-ab fragment was isolated in a single step via a His(6)-tag, which also served for its recognition by a nickel chelate-alkaline phosphatase conjugate. Thus, the recombinant F-ab fragment 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 its V-H domain alone can be readily isolated from E. coli as a soluble protein. This unusual behaviour may be explained by the 18 amino acid-long CDR-H3 and could be of value in the design of 'single domain' antibodies. (C) 1997 Federation of European Biochemical Societies.