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 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.
- MeSH
- DNA vazebné proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- endopeptidasy biosyntéza chemie genetika izolace a purifikace MeSH
- Escherichia coli enzymologie MeSH
- Haemophilus influenzae enzymologie MeSH
- kinetika MeSH
- lipidové dvojvrstvy chemie MeSH
- membránové proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- molekulární biologie metody MeSH
- proteiny z Escherichia coli biosyntéza chemie genetika izolace a purifikace MeSH
- Providencia enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- alosterická regulace * MeSH
- buněčná membrána enzymologie MeSH
- Escherichia coli enzymologie MeSH
- Haemophilus influenzae enzymologie MeSH
- membránové proteiny metabolismus MeSH
- Providencia enzymologie MeSH
- serinové proteasy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- komentáře MeSH
- práce podpořená grantem MeSH
