title: Construction, purification, and characterization of a chimeric TH1 antagonist.
abstract: BACKGROUND
  TH1 immune response antagonism is a desirable approach to mitigate some autoimmune and inflammatory reactions during the course of several diseases where IL-2 and IFN-gamma are two central players. Therefore, the neutralization of both cytokines could provide beneficial effects in patients suffering from autoimmune or inflammatory illnesses.
  
  
  RESULTS
  A chimeric antagonist that can antagonize the action of TH1 immunity mediators, IFN-gamma and IL-2, was designed, engineered, expressed in E. coli, purified and evaluated for its in vitro biological activities. The TH1 antagonist molecule consists of the extracellular region for the human IFNgamma receptor chain 1 fused by a four-aminoacid linker peptide to human 60 N-terminal aminoacid residues of IL-2. The corresponding gene fragments were isolated by RT-PCR and cloned in the pTPV-1 vector. E. coli (W3110 strain) was transformed with this vector. The chimeric protein was expressed at high level as inclusion bodies. The protein was partially purified by pelleting and washing. It was then solubilized with strong denaturant and finally refolded by gel filtration. In vitro biological activity of chimera was demonstrated by inhibition of IFN-gamma-dependent HLA-DR expression in Colo 205 cells, inhibition of IFN-gamma antiproliferative effect on HEp-2 cells, and by a bidirectional effect in assays for IL-2 T-cell dependent proliferation: agonism in the absence versus inhibition in the presence of IL-2.
  
  
  CONCLUSIONS
  TH1 antagonist is a chimeric protein that inhibits the in vitro biological activities of human IFN-gamma, and is a partial agonist/antagonist of human IL-2. With these attributes, the chimera has the potential to offer a new opportunity for the treatment of autoimmune and inflammatory diseases.
authors.affiliation: Clinical Trial Department, Center for Biological Research, Calle 134 entre 23 y 25, Cubanacan, P,O, Box 6332, Havana, Cuba. iraldo.bello@cigb.edu.cu
authors.forename: Iraldo
authors.initials: I
authors.lastname: Bello-Rivero
authors.forename: Yeny
authors.initials: Y
authors.lastname: Torrez-Ruiz
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Blanco-Garcés
authors.forename: Giselle
authors.initials: G
authors.lastname: Pentón-Rol
authors.forename: Osmani
authors.initials: O
authors.lastname: Fernández-Batista
authors.forename: Luís
authors.initials: L
authors.lastname: Javier-González
authors.forename: Haydee
authors.initials: H
authors.lastname: Gerónimo-Perez
authors.forename: Pedro
authors.initials: P
authors.lastname: López-Saura
mesh_terms: D000595:Amino Acid Sequence; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000037:antagonists & inhibitors*; D007376:Interleukin-2 / Q000819:agonists / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization; D018417:Th1 Cells / Q000187:drug effects* / Q000276:immunology
chemical_list: D000893:Anti-Inflammatory Agents; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; C511929:chimeric TH1 antagonist; D007371:Interferon-gamma