Zur Entwicklung der Homogenitaet und Homogenitaetspruefung sowie der Zaehigkeit von Generator- und Turbinenwellen auf dem europaeischen Kontinent

TIB: RN 4656 (10) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes

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