High Density Fermentation Of IL-1Ra In P.pastoris And ITS Separation, Purification And Biological Function

Interleukin 1 receptor antagonist(IL-1Ra), a natural receptor antagonist, combines with IL-1 receptor but not induce signal transduction. As a result, IL-1Ra inhibits interleukin 1(IL-1) from combining with IL-1 receptor, and IL-1Ra reduces side reaction produced by IL-1 overexpression. The article studies heterologous expression conditions of recombinant human interleukin 1 receptor antagonist( rh IL-1Ra) under different scales in Pichia pastoris(P.pastoris), separation and purification process and its biological function.Firstly, the paper studied the effects of different temperature and p H on production of rh IL-1Ra in flask. According to the result, the productivity of rh IL-1Ra was the highest when Ppastoris was induced at p H 5.5, 28 ℃. The yields of two forms of rh IL-1Ra( glycosylated and non-glycosylated) were 12 and 10 mg/L.Secondly, based on the shake-flask conditions,the optimal condition for production in 3-L bioreactor was studied, including initial inducing cell density, dissolved oxygen, temperature, p H, methanol concentration and inducing time. Then the best condition was determined, the start inducing cell optical density was 600, 20% dissolved oxygen, induced at 28 ℃, p H 5.0, for 6 d, 0.25% methanol. Under the best condition, yields of the glycosylated and non-glycosylated rh IL-1Ra were 90, 110 mg/L.Furthermore, the purification process of rh IL-1Ra was studied in our research. The best process of the separation and purification was determined, it included low-temperature centrifugal, aqueous two phase system extraction, ultra-filtration, DEAE ion exchange chromatography, SP cation-exchange chromatography. The purity of the rh IL-1Ra was more than 95%, and the recovery yield of glycosylation and non-glycosylated rh IL-1Ra reached 56% and 40%.Finally, this paper studied the function of the glycosylated and non-glycosylated rh IL-1Ra to inhibiting the rh IL-1β induced B16 apoptosis by MTT and Hoechst staining method. According to the result, the more rh IL-1Ra concentration is rising, the more obvious antagonism. And there is no significant difference between glycosylated and non-glycosylated rh IL-1Ra.In summary, this article studied the the scale of 3L expression of rh IL-1Ra in P.pastoris, purification and biological function of rh IL-1Ra, and the difference of biological effect between glycosylated and non-glycosylated rh IL-1Ra was first studied. The production of both glycosylated and non-glycosylated rh IL-1Ra in a 3L-reactor has reached a quite high level(90 and 110 mg/L). The separation and purification process of glycosylated and non-glycosylated rh IL-1Ra was established, obtaining the purity of more than 95%, the yield reached 56% and 40%. The biological differences between glycosylated and non-glycosylated of rh IL-1Ra was measured and analyzed, two kinds of rh IL-1Ra have the antagonism effect for rh IL-1β which promotes B16 cell’s apoptosis, and there is no significant difference of activities between them. This study provides a theoretical support for the study of mechanism of rh IL-1Ra, and makes foundation for the further industry research.