Production of Recombinant Rhomboid Proteases

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F17%3A00483405" target="_blank" >RIV/61388963:_____/17:00483405 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1016/bs.mie.2016.10.031" target="_blank" >http://dx.doi.org/10.1016/bs.mie.2016.10.031</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/bs.mie.2016.10.031" target="_blank" >10.1016/bs.mie.2016.10.031</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Production of Recombinant Rhomboid Proteases

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Production of Recombinant Rhomboid Proteases

Popis výsledku anglicky

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.

Druh

C - Kapitola v odborné knize

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2017

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název knihy nebo sborníku

Enzymology at the Membrane Interface: Intramembrane Proteases

ISBN

978-0-12-812213-6

Počet stran výsledku

24

Strana od-do

255-278

Počet stran knihy

474

Název nakladatele

Academic Press

Místo vydání

Cambridge

Kód UT WoS kapitoly

000403271000011