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 2.6mg enNTS1 per liter of expression culture containing 50mg of 13CH3-methionine. We also provide a 3-step purification protocol that produces final yields of 0.6mg of functional Apo-state enNTS1.

Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call