0483405 - ÚOCHB 2018 RIV US eng M - Část monografie knihy
Arutyunova, E. - Panigrahi, R. - Stříšovský, Kvido - Lemieux, M. J.
Production of Recombinant Rhomboid Proteases.
Enzymology at the Membrane Interface: Intramembrane Proteases. Cambridge: Academic Press, 2017 - (Gelb, M.), s. 255-278. Methods in Enzymology, 584. ISBN 978-0-12-812213-6
Grant CEP: GA MŠMT(CZ) LK11206; GA MŠMT LO1302
GRANT EU: European Commission(XE) 304154 - Rhomboid substrates
Grant ostatní: EMBO(DE) 2329
Institucionální podpora: RVO:61388963
Klíčová slova: rhomboid protease * ecGlpG * hiGlpG * AarA
Obor OECD: Biochemistry and molecular biology
DOI: https://doi.org/10.1016/bs.mie.2016.10.031

Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5 mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.
Trvalý link: http://hdl.handle.net/11104/0278736