IGEM:Stanford/2009/GCPR

Comments and Discussion

• Can we identify a specific GPCR or class of GPCRs that we are interested in manipulating?
• Which host organism would make the best chassis?
• What should our GPCR be sensitive to?

Papers

• Troubleshooting?? Genetic analysis of G protein-coupled receptor expression in Escherichia coli: Inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor," article from Biotechnology and Bioengineering http://www3.interscience.wiley.com/search/allsearch?mode=viewselected&product=journal&ID=121385939&view_selected.x=100&view_selected.y=6&view_selected=view_selected
• "Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli," article from Protein Science, 2008: http://www.ncbi.nlm.nih.gov/pubmed/18593817
  • Article that discusses some of the complications that emerge when expressing GPCRs in microbial systems. One of the solutions this group found was co-expression of the GPCR-GFP construct with various regulatory proteins involved in membrane metabolism. Coexpression with the AAA+ protease FtsH, an enzyme involvedin MP degradation and quality control. (Article to be read in advance of Monday's meeting)
• "High-level production, solubilization and purification of synthetic human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1." If we're still interested in veering off towards a medical application, the following article discusses success in expressing various chemokine receptors (CXC motif) in E. coli. These receptors are involved int the immune response, and have implicated in HIV infection and tumor metastasis: http://www.ncbi.nlm.nih.gov/pubmed/19223978?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
• "Characterization of Engineered Channelrhodopsin Variants with Improved Properties and Kinetics," article from Biophysical Journal, March 2009: http://www.cell.com/biophysj/fulltext/S0006-3495(09)00016-2
• Engineering G protein-coupled receptor expression in bacteria, article from PNAS, Sept. 2008: http://www.pnas.org/content/105/39/14747.full
• Database of Membrane Protein Structures: http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
• Bible of GPCR mechanisms.
• GPCR Engineered in Yeast
• The grad students in CSB 220 heard about these modified GPCRs known as RASSL (receptors adapted to synthetic ligands). May be the techniques we are looking for. Review. Methods. It doesn't seem as if this technique seems to have expanded to synbio.

Meeting Notes

• 5/4 Small Group
  • Methods/Procedures we should familiarize ourselves with: FACS, SDS-PAGE, Western-Blotting, iron-affinity chromatography
  • The paper underscored the difficulty of expressing functional GPCRs in E. coli. Can we find other papers that have troubleshooted this? Or perhaps should we designate a sub-group to examine the activity/functionality of our GPCR while the bulk of the team works on co-expressing two GPCRs in E. coli?
  • Should we consider yeast as our chassis? What are the tradeoffs? What has been done to express GPCRs in yeast?
  • Is there necessarily a trade-off between activity and modularity? (i.e. Will the time constraint limit us to either pursuing a functional GPCR or a modular and expressed but dysfunctional GPCR plasmid?)

Questions

• I would highly recommend looking through George Georgiou's literature to see what engineered parts he has made. - Chris
• Thanks for the suggestion; the article for Monday's meeting is by Georgiou. Any other recommendations?