人 DNA 连接酶 IIIα 复合物在昆虫细胞中表达后的纯化和表征。
Purification and Characterization of Human DNA Ligase IIIα Complexes After Expression in Insect Cells.
机构信息
Departments of Internal Medicine, Molecular Genetics and Microbiology and the University of New Mexico Comprehensive Cancer Center, University of New Mexico, Albuquerque, NM, USA.
Biological Systems and Engineering Division, Department of BioEngineering & BioMedical Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
出版信息
Methods Mol Biol. 2022;2444:243-269. doi: 10.1007/978-1-0716-2063-2_15.
With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to express heterologous genes in cultured insect cells has advantages for the expression of human protein complexes because of the ease of co-expressing multiple proteins in insect cells and the presence of a conserved post-translational machinery that introduces many of the same modifications found in human cells. Here we describe the preparation of recombinant baculoviruses expressing DNA ligase IIIα, XRCC1, and TDP1, their subsequent co-expression in cultured insect cells, the purification of complexes containing DNA ligase IIIα from insect cell lysates, and their characterization by multi-angle light scattering linked to size exclusion chromatography and negative stain electron microscopy.
随着生物物理方法的改进，人们对表征大型、灵活的多蛋白复合物越来越感兴趣。使用重组杆状病毒在培养的昆虫细胞中表达异源基因，对于表达人类蛋白复合物具有优势，因为在昆虫细胞中易于共表达多种蛋白，并且存在保守的翻译后修饰机制，该机制引入了许多在人类细胞中发现的相同修饰。在这里，我们描述了表达 DNA 连接酶 IIIα、XRCC1 和 TDP1 的重组杆状病毒的制备方法，它们在培养的昆虫细胞中的随后共表达，以及从昆虫细胞裂解物中纯化含有 DNA 连接酶 IIIα的复合物的方法，以及通过多角度光散射与尺寸排阻色谱和负染色电子显微镜对其进行的表征。
Zotero 插件