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

• DOI: 10.1007/978-1-4939-9121-1_3
• Publication Date: 2019-04-10T16:17:54Z
• Authors: Fabian Bumbak; Ross A. D. Bathgate; Daniel J. Scott; Paul R. Gooley
• Keywords: Neurotensin receptor; Cloning (programming)

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.