Expression and Purification of a Functional E. coli ¹³CH₃-Methionine-Labeled Thermostable Neurotensin Receptor 1...

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.