Expression and Purification of a Functional E. coli 13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies

Methods Mol Biol. 2019:1947:31-55. doi: 10.1007/978-1-4939-9121-1_3.

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 mg enNTS1 per liter of expression culture containing 50 mg of 13CH3-methionine. We also provide a 3-step purification protocol that produces final yields of 0.6 mg of functional Apo-state enNTS1.

Keywords: 13CH3-methionine; Apo-state; E. coli; GPCR; Membrane protein; Neurotensin receptor 1; Thermostable.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbon Isotopes / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Humans
  • Magnetic Resonance Spectroscopy / methods*
  • Methionine / chemistry*
  • Mutation
  • Receptors, Neurotensin / genetics
  • Receptors, Neurotensin / isolation & purification*
  • Receptors, Neurotensin / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification*
  • Recombinant Fusion Proteins / metabolism*

Substances

  • Carbon Isotopes
  • Receptors, Neurotensin
  • Recombinant Fusion Proteins
  • neurotensin type 1 receptor
  • Methionine