ABSTRACT
A number of hybrid proteins containing amino acid sequences of human lymphotoxin and human and mouse tumor necrosis factors were obtained by means of genetic engineering. By using these proteins, an antigenic determinant in the molecule of human tumor necrosis factor for monoclonal antibodies E7H2 (MAb E7H2), previously developed against this factor was localized. It was demonstrated by Western blot analysis that the MAb E7H2-binding site is located in the sequence region 37-49 of human tumor necrosis factor and includes the sequence Val41GluLeuArg44 directly interacting with MAb E7H2.
Subject(s)
Epitopes/immunology , Recombinant Fusion Proteins/immunology , Tumor Necrosis Factor-alpha/immunology , Animals , Antibody Specificity , Epitope Mapping , Genetic Engineering , Humans , Mice , Mutation , Recombinant Fusion Proteins/geneticsABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/metabolism , Recombinant Proteins/immunology , Tumor Necrosis Factor-alpha/immunology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Epitopes , Escherichia coli/genetics , Humans , Mice , Molecular Sequence Data , Mutation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Using polymerase chain reaction, a number of mutant genes encoding human tumor necrosis factor (TNF-alpha) with amino acid substitutions and a deletion were obtained. The mutant proteins (muteins) contained point mutations R32H, A33S, F144L, I118M, and I118A; double mutation R32H-F144L; and deletion of four amino acid residues 67-70. The mutant genes were expressed in E. coli under the control of constitutive promoters. A simple purification method for the muteins was developed and their physicochemical properties were studied. All the muteins obtained, except F144L and I118A, were shown by CD and cross-linking to from a spatial structure similar to that of the native TNF-alpha. The collection of muteins was characterized by their biological activity. Mutants R32H and A33S exerted a decreased cytotoxicity against murine fibroblast cell line L929, whereas point mutant F144L and double mutant R32H-F144L were essentially inactive.
