Neurotensin (NT) is a tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. Neurotensin receptor 1 (NTS1), a high affinity receptor for NT, is a member of the GPCR superfamily and is a putative target for the treatment of conditions such as Schizophrenia, Parkinson’s Disease and drug addiction. Overexpression and purification are typically limiting steps in the high resolution structure determination of GPCRs. In this study, through the optimisation of the E.coli strain used for overexpression of rat NTS1 (NTS1) and the inclusion of phospholipids in the purification buffers to prevent delipidation, an approximate 3-fold improvement in active receptor yield was obtained relative to existing protocols. Preliminary electron microscopy (negative stain and cryo) confirmed a monodisperse receptor population. Purified NTS1 is now being produced at a sufficient level for high resolution structural studies, including 3D crystallisation and further electron microscopy studies. The existing construct for the expression of NTS1 in E.coli, termed NTS1B, was modified to contain a fusion to the genes encoding either the eCFP or eYFP fluorescent proteins. These constructs were used for the E.coli expression of NTS1 tagged with either fluorescent protein at the C-terminus. Tagged receptor was successfully expressed at levels of up to 0.29 ± 0.03 mg per l of culture. Successful purification and proteolytic removal of the MBP and TrxA-His10 fusion partners was achieved whilst retaining both fluorescence and ligand binding capability (K<sub>d</sub> = 0.91 ± 0.17 nM). Purified, fluorescent receptor was reconstituted into brain polar lipid (BPL) liposomes in an active conformation which was both fluorescent and able to bind NT. Experimentation with alternative lipid compositions suggested that specific lipids are required in order to maintain ligand-binding activity. FRET between the eCFP- and eYFP-tagged receptors was observed in reconstituted samples. The FRET efficiency was comparable to that observed in vivo for other GPCRs, including the yeast α-factor receptor, which is believed to be dimeric. This suggests that NTS1 could also be multimeric. In contrast, no FRET was observed in detergent samples. Therefore, a functioning in vitro system has been developed which enables the study of NTS1 multimerisation in lipid bilayers and future studies will attempt to implement single molecule fluorescence techniques. In addition, fluorescent derivatives of NT were successfully synthesised and purified. Radioligand competition assays and fluorescence correlation spectroscopy (FCS) confirmed that the fluorescent peptides bound to purified NTS1 in specific competition with unlabelled NT. Surface plasmon resonance (SPR) was used to confirm the ligand binding activity of purified NTS1. A novel approach was utilised which involved the measurement of the binding of detergent-solubilised NTS1 to immobilised, N-terminally biotinylated NT on the sensor surface. The use of a rigorous control, which consisted of immobilised ‘scrambled sequence’ NT, demonstrated a specific interaction. Analysis of the kinetics revealed a multiphasic interaction with a K<sub>d</sub> in the nanomolar range. In summary, improvements to the expression and purification of NTS1, the generation of fluorescent constructs as useful tools in the study of receptor multimerisation and the optimisation of lipid-reconstitution protocols have opened up several preliminary lines of study which show considerable potential for future research.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:491514 |
| Date | January 2007 |
| Creators | Harding, Peter J. |
| Contributors | Watts, Anthony |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:0e84f351-696f-42c9-b2fd-8afcde6d586e |
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