Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.
The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed.
Fuchs
P
P
German Cancer Research Center, Heidelberg, Germany.
Breitling
F
F
Little
M
M
Dübel
S
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D008969:Molecular Sequence Data; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D015183:Restriction Mapping
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; C055966:immunoglobulin Fv