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

Abstract Recombinant human interferon β-1b (rhIFN β-1b) 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 β-1b IBs: 1) refining the rhIFN β-1b 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 β-1b 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 β-1b IBs. As a result, refolding of rhIFN β-1b IBs with optimal additives was under 320 MPa for 16 h, almost 80 % refolding yield was achieved even with an initial rhIFN β-1b 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 β-1b was achieved. The resulted rhIFN β-1b showed similar structures and comparable in vitro bioactivity to rhIFN β-1b 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.