AbstractG-protein coupled receptors are the largest family of integral membrane proteins found within the human genome. They function as receptors and modulators to a wide range of ligands and responses which are crucial for human health. GPCR study, specifically the investigation of structure and interaction to cognate ligands, is of high priority. Limitations for structural study can be traced in part, to obtaining suitable quantities of recombinant protein. We sought to address the limitations of traditional recombinant technologies by utilising an Escherichia coli based cell-free protein synthesis (CFPS) approach for production of a thermostable neurotensin receptor 1 (en2NTS₁). Initial results were promising, with a high amount (up to 2 mg/mL) of en2NTS₁ produced, that had attained correct secondary structure. Meanwhile, concurrent experiments indicated that CFPS produced en2NTS₁ showed non-competitive binding to the peptide ligand neurotensin8–13 when compared to E. coli produced en2NTS₁. ¹H-¹³C HMQC SOFAST NMR spectra were indicative of disrupted tertiary structure for CFPS produced ¹³CH₃-methionine labelled en2NTS₁. The results obtained, indicate CFPS produced en2NTS₁ is not forming a discrete tertiary structure and that further development of the CFPS technique needs to be carried out.