Optimized Fermentation Of IL-1Rα/HSA Fusion Protein Expressed In Pichia Pastoris Overexpression Strain

Interleukin-1 receptor antagonist is a specific and natural antagonist molecules, which is mainly used in the treatment of rheumatoid arthritis in clinical. Recent studies show that IL-1Ra has a significant therapeutic effect for type II diabetes. However, its small molecular weight leads to a half-life of 4-6h in vivo which is limited its clinical applications. So the development of long-acting interleukin-1 receptor antagonist becomes extremely important.Human serum albumin (HSA) is the most widely used protein carrier to extend the half-life of small-molecule in recent years. In April 15,2014, the FDA approved a albiglutide injection (Tanzeum) for the treatment of Type II Diabetes by GSK company of UK. Albiglutide is a recombinant HSA fusion protein of tolerated DPPIV GLP-1. The half-life of albiglutide can reach 4-7 days, which is suitable for intermittent administration for once a week, or even every two weeks. As the world’s first HSA fusion protein biopHarmaceuticals approved by the FDA, albiglutide will give more HSA fusion protein drugs new hope and motivation to be on the market.Using pPIC9 as vector expressed IL-1Rα and HSA fusion gene in Pichia pastoris, The fusion protein IL-1Rα/HSA was successfully expressed and the half-life was prolonged 20 times compared with IL-IRα, besides the pHarmacodynamics has been significantly improved. The fusion protein expression was only 100 mg/L in high density fermentation. Not only the demand of the late-stage clinical studies but also the economic benefits are all required to improve the expression of protein. By increasing the target gene copy number and co-expression with the chaperone, the expression of the overexpressed strain increased from 100 mg/L to 400 mg/L (shake flask level). But the expression level of overexpression strain was decreased in high-density, besides degradation of the fusion protein was very serious.The main contents of this paper is optimized the expression of the overexpression strain by Response Surface Methodology.1 Preliminary studies on the shake flask and high-density fermentationMany factors were affected the expression of protein in high cell density fermentation, such as the composition of the medium, pH, temperature, methanol feeding methods. If these factors were all tested in high cell density fermentation, it would take a lot of time. Therefore, we conducted preliminary studies in shake flask level to determine that pH, temperature, and adding a certain amount of yeast extract, peptone, biotin, surfactant to the medium have a significant impact on the expression of IL-1Rα/HSA fusion protein. Next, preliminary studies of high cell density fermentation to determine the 20% dissolved oxygen and high acceleration rate of methanol have a significant impact on the expression of IL-1Rα/HSA fusion protein. Through the above experiments we established the optimal medium composition and three factors of RSM pH, temperature and methanol adding rate.2 Response surface methodology optimized the expression of overexpressionResponse surface methodology is the most commonly used method in the optimization of fermentation. The advantage of RSM is to analyzed each level of the factors during the test,Overcoming the flaws of the orthogonal experiment which just analyze isolated test points and can not give an intuitive grapHical. So the RSM is widely used in experimental design and optimization. We got the optimized culture conditions of pH 7.0, T=29℃, Vm=4.81mL/L/h by RSM. In this optimal culture conditions, the expression of the fusion protein increased from 100 mg/L in single copy to about 1.14 g/L in overexpression strain, increased about 11 times.