Expression Of Interleukin-2 And Human Serum Albumin Fusion Protein In Chinese Hamster Ovary Cells

In previous study, the fusion protein of human serum albumin(HSA) and interleukin-2(IL-2) has been expressed in Pichia pastoris by our laboratory. IL-2 was conjugated to the N-(IL-2-HSA) or C-terminals(HSA-IL-2) of HSA and the obtained fusion proteins showed great biological activity. Moreover, half-life of fusion proteins extended to 13.24 h compared to 6.9 min of IL-2. However, some disadvantages of fusion proteins expressed in P. pastoris were found during fermentation, including proteolytic degradation, heterogeneous products and excessive glycosylation. Heterogeneous products of IL-2-HSA were hard to purify and excessive glycosylation of expressed protein may lead to decreased activity and shortening the half-life. All these disadvantages restricted the application of P. pastoris expression system in production of recombinant HSA-IL-2 and IL-2-HSA. Chinese hamster ovary(CHO) expression system has been successfully applied to produce several pharmaceutical products. Therefore, in this study, the fusion proteins of HSA-IL-2 and IL-2-HSA were expressed in CHO cells. The cell growth and expressing level of fusion protein expressing CHO cell lines were tested and the purification strategy and biological activity of fusion proteins were investigated.The genes of HSA-IL-2 and IL-2-HSA were inserted to the plasmid p MH3, respectively, and the obtained plasmids were transfected to CHO cells by electroporation. The stable CHO cell lines expressing HSA-IL-2(CHO/p MH3-HSA-IL-2/9) and IL-2-HSA(CHO/p MH3-IL-2-HSA/8) were screened by four clone selection and flask fermentation. Western blot(WB) results showed that fusion proteins have dual immunogenicity( HSA and IL-2). HSA-IL-2 and IL-2-HSA were cultured with CTLL-2 cells and the results of proliferation determined by MTT showed a high biological activity.After culturing in 3L-flask, the cell density of CHO/p MH3-HSA-IL-2/9 and CHO/p MH3-IL-2-HSA/8 cell line achieved 6.4×106 and 6.7×106 cells·m L-1, respectively. The production of HSA-IL-2 and IL-2-HSA at the end of fermentation were 138 and 118 mg·L-1, respectively. The results of SDS-PAGE and WB showed that no proteolytic degradation of both fusion proteins was found during the fermentation. The purification strategy was determined and the purified fusion proteins were obtained. The culture broth was collected by centrifugation and concentrated by microfiltration. The concentrated culture broth was then purified by Blue Sepharose 6FF affinity chromatography, Phenyl Sepharose 6FF hydrophobic chromatography and Q Sepharose 6FF ion exchange chromatography. The purity of HSA-IL-2 and IL-2-HSA were 96.5% and 95.3%, respectively. The yield of HSA-IL-2 was 17.29% and IL-2-HSA was 14.77%. After assaying the proliferation of CTLL-2 cell by MTT, the vitro special activity of HSA-IL-2 was 1.026×107 IU·mg-1 and IL-2-HSA was 8.3×106 IU·mg-1, which were equal to the biological activity of fusion proteins expressed by P. pastoris.