Determination of binding site of anti-tumour necrosis factor-alpha monoclonal antibody using hybrid and mutant proteins.
In order to map the immunogenic epitope for the monoclonal antibody E7H2 on the human tumour necrosis factor (hTNF-alpha) molecule, a number of chimeric proteins were developed by in-frame joining segments of the human genes encoding TNF-alpha and lymphotoxin (TNF-beta) as well as by coupling appropriate coding regions for human and mouse TNF-alpha. High level expression of these chimeric genes was achieved in Escherichia coli by placing the coding sequences under control of either E. coli trp-promoter or a tandem of bacteriophage T7 constitutive promoters A2 and A3. As revealed by Western blot analysis with monoclonal antibody E7H2 directed against human TNF-alpha, the region involved in the binding of this antibody includes sequence ValGluLeuArg in the N-terminal part of the TNF-alpha molecule.
Shingarova
L N
LN
Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
Sagaidak
L N
LN
Berkova
N
N
Korobko
V G
VG
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha