title: Protein disulfide isomerase-mediated cell-free assembly of recombinant interleukin-12 p40 homodimers.
abstract: Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two subunits, p35 and p40. The disulfide-linked homodimer (p40)2 has been shown to be a potent IL-12 antagonist. In the present study, the p40 subunit was refolded from Escherichia coli inclusion bodies. Formation of (p40)2 was greatly increased in a redox buffer containing reduced and oxidized glutathione, but was not significantly affected by the cosolvents urea, GdnHCl or Chaps. While protein disulfide isomerase (PDI), GroEL/ES or DnaK/J/GrpE suppressed aggregation during refolding of p40, only DnaK/J/GrpE and PDI enhanced p40 dimerization. Oxidative assembly of p40 into (p40)2 by PDI, but not suppression of aggregation, was strongly dependent on inclusion of BSA in the refolding buffer. It is concluded that both chaperone-like and disulfide isomerase effects are essential for correct folding of p40 into dimers.
authors.affiliation: Rega Institute for Medical Research, University of Leuven, Belgium.
authors.forename: E
authors.initials: E
authors.lastname: Martens
authors.forename: I
authors.initials: I
authors.lastname: Alloza
authors.forename: C J
authors.initials: CJ
authors.lastname: Scott
authors.forename: A
authors.initials: A
authors.lastname: Billiau
authors.forename: K
authors.initials: K
authors.lastname: Vandenbroeck
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D002474:Cell-Free System; D018834:Chaperonin 60 / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005978:Glutathione / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D018664:Interleukin-12 / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D018664:Interleukin-12; C063842:dnaK protein, E coli; D019704:Protein Disulfide-Isomerases; D005978:Glutathione