Wang Y Q, Zhou M, Zeng L M, Gao Q Y, Yuan X L, Li Y, Li M C
Zhejiang Provincial Key Laboratory of Pathophysiology, Department of Immunology, Ningbo University School of Medicine, Ningbo, 315211, China.
Biochemistry (Mosc). 2015 Feb;80(2):228-32. doi: 10.1134/S0006297915020091.
Interferon (IFN)-λ3, a member of the type III IFN family, is a pleiotropic cytokine that exhibits potent antiproliferative, antiviral, and immunoregulatory activities. For further functional study of IFN-λ3, we developed an efficient procedure that includes cloning, expression, and purification to obtain relatively large quantity of mouse IFN-λ3 fusion protein. The mature IFN-λ3 protein-coding region was cloned into the prokaryotic expression vector pET-44. IFN-λ3 contains a hexahistidine tag at its C-terminus. We used Ni(2+)-nitrilotriacetic acid agarose-affinity chromatography to purify the expressed soluble protein. The purified IFN-λ3 inhibited significantly IL-13 production in stimulated RAW264.7 macrophages. Our findings show that the production of soluble IFN-λ3 proteins by the pET-44 vector in Escherichia coli is a good alternative for the production of native IFN-λ3 and could be useful for the production of other IFN proteins.
干扰素（IFN）-λ3是III型干扰素家族的一员，是一种具有多种功能的细胞因子，具有强大的抗增殖、抗病毒和免疫调节活性。为了进一步研究IFN-λ3的功能，我们开发了一种高效的方法，包括克隆、表达和纯化，以获得相对大量的小鼠IFN-λ3融合蛋白。将成熟的IFN-λ3蛋白编码区克隆到原核表达载体pET-44中。IFN-λ3在其C末端含有一个六聚组氨酸标签。我们使用镍（2+）-次氮基三乙酸琼脂糖亲和层析法纯化表达的可溶性蛋白。纯化的IFN-λ3显著抑制了刺激的RAW264.7巨噬细胞中IL-13的产生。我们的研究结果表明，利用pET-44载体在大肠杆菌中生产可溶性IFN-λ3蛋白是生产天然IFN-λ3的一个很好的替代方法，并且可能对生产其他IFN蛋白有用。
