Panfair, DilrajkaurKusmierczyk, Andrew R.2019-06-072019-06-072016-12-17Panfair, D., & Kusmierczyk, A. R. (2016). Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach. Journal of visualized experiments : JoVE, (118), 54860. doi:10.3791/54860https://hdl.handle.net/1805/19561Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric β subunit rings sandwiched between two heptameric α subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome α and β subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete α-ring and one complete β-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.en-USPublisher PolicyBiochemistryIssue 118ProteasomeProtein assemblyArchaeaRecombinant proteinNon-denaturing polyacrylamide gel electrophoresisIn vivo assemblyArticle