多聚酸性氨基酸对蛋白质性质的影响  

Poly acidic amino acids sequence drives variation of protein in hydrodynamic properties

作　　者：李亚妃 武沈阳 褚文丹 周翠燕[1,2,3,4] 施清华 李文奇[1,2,3,4] LI Yafei;WU Shenyang;CHU Wendan;ZHOU Cuiyan;SHI Qinghua;LI Wenqi(Institute of Biomedicine Tsinghua University,Beijing 100084 China;National Protein Science Facility,Beijing 100084 China;School of Life Sciences Tsinghua University,Beijing 100084 China;Beijing Advanced Innovation Center for Structural Biology Tsinghua University,Beijing 100084 China;International School of Beijing,Beijing 101300 China)

机构地区：[1]清华大学生命科学与医学研究院,北京100084 [2]国家蛋白质科学研究(北京)设施清华基地,北京100084 [3]清华大学生命科学学院,北京100084 [4]清华大学结构生物学高精尖创新中心,北京100084 [5]北京市顺义国际学校,北京101300

出　　处：《生物学杂志》2023年第1期21-26,共6页Journal of Biology

基　　金：北京市结构生物学高精尖创新中心资助项目(20151551402)。

摘　　要：选取碳末端富含酸性氨基酸的拟南芥SnRK2.6(sucrose non-fermenting1-related protein kinase 2.6)和人源PDI(protein disulfide isomerase),以及近球形蛋白拟南芥PYL10 (PYR like protein 10),分别将重复酸性氨基酸序列添加到SnRK2.6(1-332)、PDI(1-440)、PYL10碳末端,利用大肠杆菌BL21重组表达,经过亲和层析,离子交换层析和分子排阻层析进行纯化,综合利用分析超速离心技术,分子排阻层析技术以及多角度静态光散射技术,研究人为设计的多聚氨基酸末端对蛋白质分子排阻行为,聚合状态和其他水力学性质的影响。结果发现,多聚酸性氨基酸末端虽不影响蛋白质分子的聚合状态,但会明显减少分子排阻色谱中蛋白质的洗脱体积,影响蛋白质分子的斯托克斯半径和轴长比等水力学性质。Here several repeats of Asp/Glu were added to the C-terminal of SnRK2.6(1-332), PDI(1-440) and PYL10, which SnRK2.6 and PDI had a natural acidic amino acids tail on their C-terminal, and PYL10 performed like a globular protein. Recombinant proteins were produced by E.coli BL21 followed by purification with affinity chromatography, ion-exchange column chromatography and size exclusion chromatography. With analytical ultracentrifugation, static light scattering analysis and size exclusion chromatography, we demonstrated that the addition of acidic amino acids on the C-terminal resulted in early elution on size exclusion chromatography without impacting oligomerization, while hydrodynamic properties such as stokes radius and friction ration increased clearly.

关 键 词：分析超速离心 多角度静态光散射 分子排阻层析 多聚酸性氨基酸 水力学性质 

分 类 号：Q51[生物学—生物化学]