Molecular Analysis of Hemolysin-Mediated Secretion of a Human interleukin-6 Fusion Protein in Salmonella Typhimurium

FEMS Immunol Med Microbiol. 2000 Apr;27(4):333-40. doi: 10.1111/j.1574-695X.2000.tb01447.x.

Abstract

Previously, we reported a plasmid-bearing Salmonella typhimurium strain capable of secreting human interleukin-6 (hIL-6) when genetically fused to the Escherichia coli hemolysin transport signal (HlyA(S)). Stationary phase culture supernatants of this strain revealed three major forms of hIL-6-HlyA(S) fusion protein (apparent molecular masses 32.4, 30.3, 27.0 kDa), at which the largest protein presumably represented full-length hIL-6-HlyA(S). The biological activity of the hIL-6-HlyA(S) protein mixture was similar to that of mature hIL-6. Accumulation of hIL-6-HlyA(S) in the culture supernatant occurred only during the initial growth phase, whereas in stationary phase and under in vitro conditions successive cleavage into the two truncated forms was observed. On the other hand, in whole cell lysates only full-length hIL-6-HlyA(S) could be detected, accounting for more than 50% of the totally synthesized protein. Upon cell fractionation, cellular hIL-6-HlyA(S) was exclusively found in the membrane fraction. These results suggest, that in S. typhimurium production and secretion of hIL-6-HlyA(S) is restricted to growing cells. A specific processing by a Salmonella-derived protease did not affect the biological activity of the fusion protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Blotting, Western
  • Cell Fractionation
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases / metabolism
  • Escherichia coli / metabolism
  • Escherichia coli Proteins*
  • Genetic Vectors
  • Hemolysin Proteins / genetics
  • Hemolysin Proteins / metabolism*
  • Humans
  • Interleukin-6 / genetics
  • Interleukin-6 / metabolism*
  • Mice
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Salmonella typhimurium / genetics*
  • Salmonella typhimurium / growth & development
  • Salmonella typhimurium / metabolism

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Hemolysin Proteins
  • Hlya protein, E coli
  • Interleukin-6
  • Recombinant Fusion Proteins
  • Endopeptidases