Efficient purification of recombinant human tumor necrosis factor beta from Escherichia coli yields biologically active protein with a trimeric structure that binds to both tumor necrosis factor receptors

J Biol Chem. 1991 Feb 25;266(6):3863-9.

Abstract

A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described. The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay. The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha. However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species. rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity. The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha.

MeSH terms

  • Binding, Competitive
  • Blotting, Western
  • Chromatography, DEAE-Cellulose
  • Chromatography, Gel
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism*
  • Gene Expression Regulation, Bacterial
  • Humans
  • Lymphotoxin-alpha / genetics
  • Lymphotoxin-alpha / isolation & purification*
  • Lymphotoxin-alpha / metabolism
  • Receptors, Cell Surface / metabolism*
  • Receptors, Tumor Necrosis Factor
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • Lymphotoxin-alpha
  • Receptors, Cell Surface
  • Receptors, Tumor Necrosis Factor
  • Recombinant Proteins