秘鲁鱿鱼肌动蛋白的分离纯化及结构初探

Isolation, Purification and Structural Exploration of Actin from Dosidicus gigas

目的:建立从秘鲁鱿鱼肌原纤维蛋白中分离纯化肌动蛋白的方法,实现肌动蛋白的批量制备。方法:使用磷酸盐缓冲液从秘鲁鱿鱼中提取肌原纤维混合蛋白,提取液首先经过阴离子交换层析分离出只有微小电荷差别的蛋白质,然后用凝胶过滤层析对混合组分中的小分子物质进行去除(如脱盐等操作),再使用大分子蛋白分离纯化系统纯化,得到纯度高的肌动蛋白(AC)。采用SDS-PAGE、HPLC、MALDI-TOF-MS 3种方法鉴定所分离的蛋白纯度和分子质量,采用光散射技术对肌动蛋白在溶液中的粒径分布进行表征。结果:本文从肌原纤维蛋白中提取出了纯度较高的肌动蛋白,且肌动蛋白的水力学半径(Rh)为3.46 nm。结论:初步建立一种分离纯化肌原纤维蛋白中AC的新方法,为实现肌动蛋白的批量制备提供技术支撑。

Objective:To establish a method for isolating and purifying actin from Dosidicus gigas myofibrillary protein and realize the batch preparation of actin.Method:The mixed protein of myofibrillar was extracted from Dosidicus gigas by phosphate buffer.The protein extracted from the squid was separated by anion exchange chromatography with only a small charge difference.Then the gel filtration chromatography was used to remove the small molecules in the mixture,such as desalination,and then purified by macromolecular protein purification.The purified protein was obtained with high purity(AC).SDS-PAGE,HPLC and MALDI-TOF-MS were used to identify the purity and molecular weight of the isolated protein.The particle size distribution of actin in the solution was characterized by light scattering technique.Results:In this paper,actin with high purity was extracted from myofibrillar protein,and the hydraulic radius(Rh)of actin was 3.46 nm.Conclusion:A new method for isolation and purification of actin in myofibrillar protein was preliminarily established,which provided support for the batch preparation of actin.