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 - PubMed (original) (raw)
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
W Schiweck et al. FEBS Lett. 1997.
Free article
Abstract
The cDNAs for the two variable domains of the antibody 9E10 were cloned from the hybridoma cell line. A chimeric 9E10 Fab fragment was produced in E. coli under control of the tightly controlled tetracycline promoter. The functional Fab fragment was isolated in a single step via a His6-tag, which also served for its recognition by a nickel chelate-alkaline phosphatase conjugate. Thus, the recombinant Fab 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 Fv 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.
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