High hydrostatic pressure refolding of highly hydrophobic protein: A case study of recombinant human interferon β-1b from inclusion bodies

Recombinant human interferon beta-lb (rhIFN beta-lb) represents a highly hydrophobic protein and is difficult to refold from inclusion bodies (IBs) using conventional methods. In this paper, we report a strategy of high hydrostatic pressure (HHP) combining with additives for facile refolding of rhIFN beta-lb IBs: 1) refining the rhIFN beta-lb IBs with 8 M urea supplemented with 1% Triton X-100 to reduce the amount of impurities; 2) adding 5 mM DTT to enable 100 % mass yield of rhIFN beta-lb IBs; 3) addition of zwittergent 3-14 could significantly enhance the initial refolding concentration while did not disturb the following purification; 4) maintaining a pressure of 320 MPa could ensure considerable refolding efficacy of rhIFN beta-lb IBs. As a result, refolding of rhIFN beta-lb IBs with optimal additives was under 320 MPa for 16 h, almost 80 % refolding yield was achieved even with an initial rhIFN beta-lb IBs suspension of 4 mg/mL. After purification by SP-Sepharose fast flow (SP FF) and Butyl-S fast flow (Butyl-S FF), an overall yield of 37.8 % of rhIFN beta-lb was achieved. The resulted rhIFN beta-lb showed similar structures and comparable in vitro bioactivity to rhIFN beta-lb standard. The whole process featured integration of solubilization/refolding with high initial refolding concentration and considerable yield, indicating HHP refolding is a promising strategy for production of highly hydrophobic proteins from IBs.

Automated summary

The study successfully refolded the highly hydrophobic protein rhIFN beta-lb IBs using a strategy of high hydrostatic pressure combined with additives, achieving a good yield. The resulting product showed similar structures and in vitro bioactivity to the standard product.