ArticleMethods in molecular biology (Clifton, N.J.)2018
Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes.
Article in Methods in molecular biology (Clifton, N.J.), 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 23 citations in OpenAlex.
- Mapping Interaction of Assembly Factor Rpn14 With the Proteasome Base Reveals a Bipartite Interface and Implies Ordered Remodeling of Intersubunit Contacts During Proteasome Biogenesis.Journal of molecular biology · 2026Article
- Identifying endogenous substrates of the 26S proteasome through site-specific photocrosslinking.bioRxiv : the preprint server for biology · 2026Article
- Faf1 accelerates p97-mediated protein unfolding by promoting ubiquitin engagement.Cell reports · 2026Article
- Substrate-interacting pore loops of two ATPase subunits determine the degradation efficiency of the 26S proteasome.Nature communications · 2026Article
- Faf1 accelerates p97-mediated protein unfolding by promoting ubiquitin engagement.bioRxiv : the preprint server for biology · 2025Article
- The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome.Cell reports · 2025Article
- Recombinant Expression of Photo-crosslinkable 26S Proteasome Base Subcomplex.bioRxiv : the preprint server for biology · 2024Article
- Non-Canonical Amino Acids in Analyses of Protease Structure and Function.International journal of molecular sciences · 2023Review
- Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2.eLife · 2023Article
- The Ufd1 cofactor determines the linkage specificity of polyubiquitin chain engagement by the AAA+ ATPase Cdc48.Molecular cell · 2023Article
- High-Throughput Assay for Characterizing Rpn11 Deubiquitinase Activity.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Ubiquitin modulates 26Science advances · 2022Article
- Proteasomal conformation controls unfolding ability.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
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Corrections and comments
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Authors and funding
2 authors at 2 institutions in 1 country.
Funding
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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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.