A Hybrid G-Protein Coupled Neurotensin Receptor (NTS1) with Wild Type EnvZ expressed in E.coli (BSB)

P F LoCascio et al.
University of Oxford

How do you choose chimeric pieces? Look for generalizable characteristics, e.g. mechanical transduction of a signal. They chose EnvZ-OmpR from E.coli. This is a generic system: phosphorylation triggered depending on the amount of osmotic pressure. They end up with a simple custom-designed biological circuit. Components are essentially isolated, so you can change one component at a time. They used the internal part with a different external component (the transmembrane response to osmotic pressure change). They used a conserved proline to change what was at the front of the construct.

Why a GPCR? They are a large class of pharmaceutical targets (>30%), and a working GPCR in a microbial system could be part of a molecular sensing tool (need to couple with WR signalling mechanism). Neurotensin is a GPCR from R.Norvegicus. When designing the hybrid, they need to couple the external signal with the internal transducer to activate the internal transcriptional response. HAMP domain needs a mechanical stimulus from linker peptid, to activate auto-phosphorylation. They know that the GPCR has some sort of twisting motion, but the 3d structure is not yet known. GPCRs have 7 transmembrane helices. There is an interesting "8th half-helix" sitting on the end of the 7th helix.

The sequencing was correct – all plasmids were verified correct. They plan to express plasmids into the reporter system, test ligand binding, and test dimerisation effect.

Monday Session 2
http://friendfeed.com/rooms/biosysbio
http://conferences.theiet.org/biosysbio

Please note that this post is merely my notes on the presentation. They are not guaranteed to be correct, and unless explicitly stated are not my opinions. They do not reflect the opinions of my employers. Any errors you can happily assume to be mine and no-one else's. I'm happy to correct any errors you may spot – just let me know!

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