Generation and characterization of soluble interleukin‐33 receptor fused with immunoglobulin gamma‐1 constant domain expressed by P ichia pastoris yeast

Objectives Interleukin ( IL )‐33 is a novel member of pro‐inflammatory cytokine IL ‐1 family, which plays an important role in the immune response. IL ‐33 was proved to involve in many inflammatory and allergic diseases, thus the inhibition of this cytokine may be a promising treatment for these diseases. Arms of the study were to generate mouse soluble IL ‐33 receptor fused with human IgG1 F c domain (ms IL 33 R ‐ F c) expressed by P ichia pastoris yeast and to characterize the IL ‐33 inhibitory activity of this protein. Methods Clone of P . pastoris expressing ms IL 33 R ‐ F c was established and the recombinant protein was harvested from culture supernatant by protein A sepharose beads. Recombinant msIL33R ‐ Fc was analysed by SDS ‐ PAGE and Western blotting and activity of the protein was investigated using the immunoprecipitation and the bio‐assay on EL ‐4 cells. Key findings P . pastoris ‐derived ms IL 33 R ‐ F c was expressed as a glyco‐protein and perhaps in dimeric form. The glycosylation of protein expressed by P . pastoris yeast was more intensive and more heterogeneous compared with the counterpart protein expressed from HEK 293 cells. Similar to HEK 293‐derived protein, ms IL 33 R ‐ F c from P . pastoris was able to capture IL ‐33 and to interfere with the interaction between IL ‐33 and IL ‐33 R in in‐vitro condition. In IL ‐33‐stimulated EL ‐4 cell bio‐assay, P . pastoris ‐derived ms IL 33 R ‐ F c suppressed IL ‐33 activity similarly as HEK 293‐derived ms IL 33 R ‐ F c did.Conclusions P . pastoris yeast can express and secrete bio‐functional fusion protein sIL33R‐Fc IgG 1 and this expression system may be beneficial in future studies on the fusion protein.