细胞因子人脂联素全序列蛋白的真核表达及晶体生长

Eukaryotic Expression and Crystal Growth of a Cytokines:Full-sequence Adiponectin

目的:找寻适用于脂联素全序列蛋白的结晶条件,为解析其空间结构奠定基础,从而研究脂联素聚合体的内在构成模式,为开发高活性脂联素类衍生细胞因子提供参考。方法:首先构建脂联素全序列蛋白的真核表达载体,对其进行诱导表达,然后通过经亲和层析和凝胶过滤分离纯化后,得到高纯度的脂联素全序列蛋白,最后尝试使用坐滴法和悬滴法以及多种温度环境和结晶液条件,从而找寻适于脂联素全序列蛋白质的结晶条件。结果:通过纯化后的脂联素蛋白纯度可以达到91.3%,在溶液中的粒径分布于2 nm到4 nm。在线性变温条件下(24 h内,由277 K线性升温至313 K,再线性降温至277 K),通过悬滴法于48 h可获得脂联素全序列蛋白的针状晶体。结论:本研究选择真核载体,以亲和层析和凝胶过滤为分离纯化手段,得到了纯度高,粒径均一的脂联素全序列蛋白。随后通过尝试多种结晶方法、条件和环境,初步确定获得脂联素全序列蛋白晶体的条件,为后续获得高质量单晶提供了参考。

Objective： To find the suitable crystallization conditions of full-sequence adiponectin so as to laying a foundation for future study of its crystal structure, the inherent structure model of polymer, and higher active derivatives. Methods： First, the eukaryotic expression vector of full-sequence adiponectin protein was constructed and induced. Second, high purity protein was get through the affinity chromatography and gel filtration purification. At last, the suitable crystallization conditions of adiponectin was obtained by a series of test, such as sitting drop method, hanging drop method as well as a variety of environmental and crystallization conditions. Results： After purification, the protein purity of adiponectin reached 91.3 %, and it＇s particle size distributed in the range of 2 nm to 4 nm. Under the linear temperature program （the temperature was increased linearly from 277 K to 313 K over time 12 h and decrease linearly from 313 K to 277 K over the same, and then hold on 277 K）, the acicular crystals of full-sequences adiponectin can be obtained by the hanging drop method in 48 h. Conclusions： In this study, eukaryote was chosen to be the host and the high purity full-sequences adiponectin protein was obtained by affinity chromatography and gel filtration purification method. And then, the crystallization conditions of adiponectin were preliminary got by trying a variety of crystallization method and the conditions, which provided a reference for the subsequent study of high quality single crystal.