Abstract: Interferon-kappa (IFN-κ) has important biofunctions such as antivirus, antitumor, and immunomodulation. The unique secretion and physiological characteristics make it a promising medicine in clinical treatments. The recombinant human interferon-kappa (rhIFN-κ) expressed as inclusion bodies in E. coli require in vitro refolding to restore its biological functions, yet it tends to form precipitates in the process. Moreover, IFN-κ contains two disulfide bonds, but they are difficult to be correctly oxidized during the normal refolding procedure. In this work, a strong anionic surfactant, sodium dodecyl sulfate (SDS), was used to solubilize the inclusion bodies, and polyol 2-methyl-2,4-pentanediol (MPD) was added to the refolding buffer to gradually strip the SDS from the protein during the renaturation process to complete the refolding of rhIFN-κ. Experimentation showed that identified by the oxidative degree of its two disulfide bonds, there were mainly three species of rhIFN-κ after refolding: the completely oxidated rhIFN-κ, partially oxidated intermediate, and unoxidized species. For the SDS/MPD refolding system, the refolding yield for the correctly oxidated rhIFN-κ was closely related to the concentration and ratio of SDS and MPD in the renaturation system. Insufficient SDS or excessive MPD might result in forming aggregates or precipitates, while the correct oxidization of the disulfide bond would be suppressed at high SDS/MPD concentration ratios. Upon further optimization of the redox system and the solution pH, the optimal buffer was found by adding 0.05wt% SDS, 1 mol/L MPD, 0.2 mmol/L GSSG, and 0.1 mmol/L GSH to 20 mmol/L Tris at pH=9.5. Moreover, increasing protein concentration to 2.0 mg/mL would not significantly decrease the refolding yield. Under the optimized condition, the refolding yield could reach 66% after incubation for 24 hours at room temperature. Further purification with reversed-phase liquid chromatography could effectively remove the misfolded species as well as other impurities, achieving purity of 90% rhIFN-κ with only one band in non-reducing SDS-PAGE. The results showed that the SDS/MPD system could effectively suppress aggregation while increasing the rate of correct oxidization of the disulfide bonds. Our study lay a solid foundation for the production and clinical applications for rhIFN-κ in the future.

Key words: rhIFN-κ, inclusion body, disulfide bond oxidation, SDS/MPD refolding system

摘要： 干扰素卡帕(Interferon-Kappa, IFN-κ)是一种具有抗病毒、抗肿瘤及免疫调节等重要功能的蛋白质，独特的分泌方式和生理功能使其在临床上具有良好的应用前景。通过大肠杆菌以包涵体形式表达的重组人干扰素卡帕(rhIFN-κ)需要经过体外折叠复性才能获得生物活性，由于IFN-κ含两对二硫键，因此复性时面临易生成沉淀和二硫键难以正确氧化等双重挑战。本研究采用强阴离子表面活性剂十二烷基硫酸钠(SDS)溶解包涵体，并在复性过程添加多元醇2-甲基-2,4-戊二醇(MPD)逐渐剥离与蛋白质紧密结合的SDS，实现rhIFN-κ的氧化复性。研究发现，rhIFN-κ复性后主要形成了二硫键不同氧化程度的三种结构，包括完全氧化的正确结构、部分氧化的中间体和完全未氧化的变性蛋白。在SDS/MPD复性体系中，rhIFN-κ完全氧化的复性收率与两者浓度及比例密切相关。SDS浓度过低或MPD浓度过高易导致蛋白质沉淀或聚集，SDS浓度过高或MPD浓度过低则会抑制二硫键的正确氧化。进一步对氧化还原体系及pH值进行优化，确定了最优的复性缓冲液：添加0.05wt% SDS, 1 mol/L MPD, 0.2 mmol/L氧化型谷胱甘肽(GSSG), 0.1 mmol/ L还原型谷胱甘肽(GSH)的20 mmol/L Tris, pH=9.5。且在此条件下，蛋白质浓度提高至2.0 mg/mL时，rhIFN-κ复性收率基本不变。室温复性24 h，rhIFN-κ的复性收率达66%。利用制备反相高效液相色谱一步纯化，可有效去除杂质及复性中间体等错误结构，获得非还原电泳为单一条带的纯度90%的rhIFN-κ。结果表明，SDS/MPD体系能抑制包涵体复性时沉淀形成的同时还能有效提高二硫键的正确氧化，该结果可为rhIFN-κ的规模化生产奠定良好的基础。

关键词: 重组人IFN-κ, 包涵体, 氧化复性, SDS/MPD复性系统