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 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.
Schiweck
W
W
Institut für Biochemie, Technische Hochschule, Darmstadt, Germany.
Buxbaum
B
B
Schätzlein
C
C
Neiss
H G
HG
Skerra
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005803:Genes, Immunoglobulin; D006639:Histidine; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D017421:Sequence Analysis; D012995:Solubility
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine