Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta.
Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes. To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E. coli strain JM105. rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC). Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained. Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta. Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments. Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained. Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC. Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay. Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts. These observations indicate that E. coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D009000:Monocytes / Q000032:analysis; D011971:Receptors, Immunologic / Q000378:metabolism; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013582:Synovial Fluid / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects
D007375:Interleukin-1; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA