Abstract
Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inhibitors.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ciechanover A (2017) Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Best Pract Res Clin Haematol 30(4):341–355. https://doi.org/10.1016/j.beha.2017.09.001
Collins GA, Goldberg AL (2017) The logic of the 26S proteasome. Cell 169(5):792–806. https://doi.org/10.1016/j.cell.2017.04.023
Budenholzer L, Cheng CL, Li Y, Hochstrasser M (2017) Proteasome structure and assembly. J Mol Biol 429(22):3500–3524. https://doi.org/10.1016/j.jmb.2017.05.027
Basler M, Kirk CJ, Groettrup M (2013) The immunoproteasome in antigen processing and other immunological functions. Curr Opin Immunol 25(1):74–80. https://doi.org/10.1016/j.coi.2012.11.004
Tenzer S, Stoltze L, Schonfisch B, Dengjel J, Muller M, Stevanovic S, Rammensee HG, Schild H (2004) Quantitative analysis of prion-protein degradation by constitutive and immuno-20S proteasomes indicates differences correlated with disease susceptibility. J Immunol 172(2):1083–1091
Lázaro S, Gamarra D, Del Val M (2015) Proteolytic enzymes involved in MHC class I antigen processing: a guerrilla army that partners with the proteasome. Mol Immunol 68(2 Pt A):72–76. https://doi.org/10.1016/j.molimm.2015.04.014
Liepe J, Marino F, Sidney J, Jeko A, Bunting DE, Sette A, Kloetzel PM, Stumpf MP, Heck AJ, Mishto M (2016) A large fraction of HLA class I ligands are proteasome-generated spliced peptides. Science 354(6310):354–358
Vigneron N, Ferrari V, Stroobant V, Abi Habib J, Van den Eynde BJ (2017) Peptide splicing by the proteasome. J Biol Chem 292(51):21170–21179. https://doi.org/10.1074/jbc.R117.807560
Stratikos E, Stern LJ (2013) Antigenic peptide trimming by ER aminopeptidases—insights from structural studies. Mol Immunol 55(3–4):212–219. https://doi.org/10.1016/j.molimm.2013.03.002
Blees A, Januliene D, Hofmann T, Koller N, Schmidt C, Trowitzsch S, Moeller A, Tampé R (2017) Structure of the human MHC-I peptide-loading complex. Nature 551(7681):525–528. https://doi.org/10.1038/nature24627
Tenzer S, Wee E, Burgevin A, Stewart-Jones G, Friis L, Lamberth K, Chang CH, Harndahl M, Weimershaus M, Gerstoft J, Akkad N, Klenerman P, Fugger L, Jones EY, McMichael AJ, Buus S, Schild H, van Endert P, Iversen AK (2009) Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance. Nat Immunol 10(6):636–646. https://doi.org/10.1038/ni.1728. [pii] ni.1728
Tenzer S, Crawford H, Pymm P, Gifford R, Sreenu VB, Weimershaus M, de Oliveira T, Burgevin A, Gerstoft J, Akkad N, Lunn D, Fugger L, Bell J, Schild H, van Endert P, Iversen AK (2014) HIV-1 adaptation to antigen processing results in population-level immune evasion and affects subtype diversification. Cell Rep 7(2):448–463. https://doi.org/10.1016/j.celrep.2014.03.031
Wang Y, Lin Y, Shu M, Wang R, Hu Y, Lin Z (2013) Proteasomal cleavage site prediction of protein antigen using BP neural network based on a new set of amino acid descriptor. J Mol Model 19(8):3045–3052. https://doi.org/10.1007/s00894-013-1827-7
Stranzl T, Larsen MV, Lundegaard C, Nielsen M (2010) NetCTLpan: pan-specific MHC class I pathway epitope predictions. Immunogenetics 62(6):357–368. https://doi.org/10.1007/s00251-010-0441-4
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft TE599/1-1, TE599/2-1 and the Forschungszentrum Immuntherapie (FZI CF7) of the University Mainz.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Tenzer, S., Schild, H. (2019). Purification of Large Cytosolic Proteases for In Vitro Assays: 20S and 26S Proteasomes. In: van Endert, P. (eds) Antigen Processing. Methods in Molecular Biology, vol 1988. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9450-2_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9450-2_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9449-6
Online ISBN: 978-1-4939-9450-2
eBook Packages: Springer Protocols