Purification and Characterization of Human DNA Ligase IIIα Complexes After Expression in Insect Cells.

Autor:	Rashid I; Departments of Internal Medicine, Molecular Genetics and Microbiology and the University of New Mexico Comprehensive Cancer Center, University of New Mexico, Albuquerque, NM, USA., Tsai MS; Biological Systems and Engineering Division, Department of BioEngineering & BioMedical Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA., Sverzhinsky A; Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, QC, Canada., Hlaing AS; Biological Systems and Engineering Division, Department of BioEngineering & BioMedical Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA., Shih B; Biological Systems and Engineering Division, Department of BioEngineering & BioMedical Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA., Thwin AC; Biological Systems and Engineering Division, Department of BioEngineering & BioMedical Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA., Lin JG; Biological Systems and Engineering Division, Department of BioEngineering & BioMedical Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA., Maw SS; Biological Systems and Engineering Division, Department of BioEngineering & BioMedical Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA., Pascal JM; Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, QC, Canada., Tomkinson AE; Departments of Internal Medicine, Molecular Genetics and Microbiology and the University of New Mexico Comprehensive Cancer Center, University of New Mexico, Albuquerque, NM, USA. atomkinson@salud.unm.edu.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2444, pp. 243-269.
DOI:	10.1007/978-1-0716-2063-2_15
Abstrakt:	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. (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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