Analysis of the structure-function relationship of tumour necrosis factor. Human/mouse chimeric TNF proteins: general properties and epitope analysis.
To analyse the structure-function relationship of tumour necrosis factor (TNF), a set of in-frame chimeric genes was constructed by coupling appropriate segments of the human and mouse TNF coding regions. Under control of the bacteriophage lambda inducible PL promoter high level expression of these chimeric genes was obtained in Escherichia coli. Although both human and mouse TNF were produced in E. coli as soluble proteins, a reduction of solubility was observed in some of the chimeric proteins. The specific activity was variable, but in some constructs comparable to human TNF, indicating that the structural conformation of these chimeric proteins resembled the human TNF structure. Neutralization analysis using two monoclonal antibodies directed against human TNF, indicated that the regions involved in the binding of these antibodies are distributed over multiple segments of the polypeptide. Further analysis by site-directed mutagenesis of one subregion allowed the identification of the Arg131 residue as involved in the binding of both neutralizing monoclonal antibodies; an Arg131----Gln replacement abolished antibody binding but did not affect the specific activity of TNF.
Laboratorium Molekulaire Biologie, Ghent, Belgium.
J
J
Tavernier
A
A
Marmenout
R
R
Bauden
G
G
Hauquier
X
X
Van Ostade
W
W
Fiers
D000595:Amino Acid Sequence; D000818:Animals; D002678:Chimera; D000939:Epitopes / Q000032:analysis*; D005796:Genes; D014644:Genetic Variation; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009500:Neutralization Tests; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology
D000939:Epitopes; D014409:Tumor Necrosis Factor-alpha