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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
Author(s) -
Schiweck Wolfram,
Buxbaum Britta,
Schätzlein Christian,
Neiss Hans Günther,
Skerra Arne
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00983-6
Subject(s) - microbiology and biotechnology , recombinant dna , chemistry , antibody , conjugate , peptide , dissociation constant , peptide sequence , stereochemistry , biochemistry , biology , gene , receptor , mathematical analysis , mathematics , immunology
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.