Analysis of human IL-6 mutants expressed in Escherichia coli. Biologic activities are not affected by deletion of amino acids 1-28.
We have constructed and analyzed amino terminally deleted analogs of IL-6. Progressively shortened variants of mature IL-6 were constructed at the cDNA level and expressed in Escherichia coli. Mutant proteins were recovered from refractile bodies by solubilizing in 6 M guanidine-HCl. The mutant protein concentration in these preparations was estimated by Western blotting by using an IL-6-specific mAb and the biologic activity was measured in the B9 (hybridoma growth factor) assay. The first 28 amino acids of mature IL-6 could be removed without significantly affecting biologic activity. A further removal of amino acids 29 and 30 resulted in an approximately 50-fold decrease, whereas removal of amino acids 31 to 34 virtually abolished the activity. The mutants showed the same reaction pattern in three other IL-6 assays: induction of murine thymocyte proliferation, induction of fibrinogen synthesis by a human hepatoma cell line (HepG2), and the induction of IgM synthesis by an EBV-transformed B cell line. This suggests that a single functional domain might be responsible for all four activities of IL-6.
Brakenhoff
J P
JP
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Hart
M
M
Aarden
L A
LA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014644:Genetic Variation; D005822:Genetic Vectors; D006801:Humans; D015850:Interleukin-6; D007378:Interleukins / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000502:physiology*
D000596:Amino Acids; D015850:Interleukin-6; D007378:Interleukins; D011994:Recombinant Proteins