@inbook{afbbbbbb2a2648efb39f18256a3957ac,
title = "Expression and Purification of a Functional E. coli 13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies",
abstract = "Escherichia coli (E. coli) is the most widely used expression host for recombinant proteins due to high expression yields and straightforward molecular cloning. Directed evolution of G protein-coupled receptors (GPCRs) has made several of these difficult to express membrane proteins amenable to prokaryotic expression. Here, we describe a protocol for near complete 13CH3-methionine labeling of a thermostable neurotensin receptor 1 (enNTS1) variant in E. coli for solution NMR-based dynamics studies. Our expression strategy utilizes methionine biosynthesis pathway inhibition forcing E. coli to incorporate exogenous methionine with 96% efficiency at expression levels of 2.6{\^A} mg enNTS1 per liter of expression culture containing 50{\^A} mg of 13CH3-methionine. We also provide a 3-step purification protocol that produces final yields of 0.6{\^A} mg of functional Apo-state enNTS1.",
keywords = "CH-methionine, Apo-state, E. coli, GPCR, Membrane protein, Neurotensin receptor 1, Thermostable",
author = "Fabian Bumbak and Bathgate, {Ross A.D.} and Scott, {Daniel J.} and Gooley, {Paul R.}",
note = "Publisher Copyright: {\textcopyright} 2019, Springer Science+Business Media, LLC, part of Springer Nature.",
year = "2019",
doi = "10.1007/978-1-4939-9121-1_3",
language = "English",
series = "Methods in Molecular Biology",
publisher = "Humana Press",
pages = "31--55",
booktitle = "Methods in Molecular Biology",
address = "United States of America",
}