Abstract
The 26S proteasome is the major regulated protease in eukaryotes and is responsible for degrading ubiquitinated substrates. It consists of a barrel-shaped 20S core peptidase and one or two 19S regulatory particles, which recognize, unfold, and translocate substrates into the core. The regulatory particle can be further divided into two multi-subunit complexes: the base and the lid. Here we present protocols for expressing the Saccharomyces cerevisiae base and lid recombinantly in Escherichia coli and purifying the assembled subcomplexes using a tandem affinity purification method. The purified complexes can then be reconstituted with 20S core to form fully functional proteasomes. Furthermore, we describe a method for incorporating the unnatural amino acid p-azido-l-phenylalanine into the recombinant complexes at any residue position, allowing for non-disruptive site-specific modifications of these large assemblies. The use of recombinant proteins allows for complete mutational control over the proteasome regulatory particle, enabling detailed studies of the mechanism by which the proteasome processes its substrates. The ability to then specifically modify residues in the regulatory particle opens the door to a wide range of previously impossible biochemical and biophysical studies. The techniques described below for incorporating unnatural amino acids into the proteasomal subcomplexes should be widely transferable to other recombinant proteins, whether individually purified or in larger multi-subunit assemblies.
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Acknowledgments
We thank the members of the Martin Lab for helpful discussions and the Prof. P. Schulz Lab at the Scripps Research Institute as well as the Prof. F. Isaacs Lab at Yale University for providing plasmid constructs for unnatural amino acid incorporation. J.A.M.B. acknowledges support from the NSF Graduate Research Fellowship. This research was funded in part by the US National Institutes of Health (R01-GM094497 to A.M.), the US National Science Foundation CAREER Program (NSF-MCB-1150288 to A.M.), and the Howard Hughes Medical Institute (A.M.).
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Bard, J.A.M., Martin, A. (2018). Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_15
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DOI: https://doi.org/10.1007/978-1-4939-8706-1_15
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