Protein disulfide isomerase-mediated cell-free assembly of recombinant interleukin-12 p40 homodimers

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 refol ded from Escherichia coli inclusion bodies. Formation of (p40)(2) was great ly 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 suppress ed aggregation during refolding of p40, only DnaK/J/GrpE and PDI enhanced p 40 dimerization. Oxidative assembly of p40 into (p40)(2) by PDI, but not su ppression of aggregation, was strongly dependent on inclusion of BSA in the refolding buffer. It is concluded that both chaperone-like and disulfide i somerase effects are essential for correct folding of p40 into dimers.