Midweek Meeting Agenda
Summary of System
- Kabat Database
- Germline Website - Antibody Anatomy e-mail
- AntiRabbit scFv in GenBank
- Other scFv in mice (5?) (monoclonal)
- Antigens (how to search?)
- Main Issue: Traversing the outer membrane and cell wall
- ToxR - dimerizing transmembrane protein that directly transcribes at its DNA binding site. Located across the inner membrane. Has already been ported into e. Coli.
- Tar/MCPs - Part of chemotaxis pathways, responds to aspartate. Inner transmembrane protein that already exists as a dimer, contrary to our initial hope. There is still a potential application.
- FecA - The only outer membrane receptor we have found. It moves Fe3+ into the cell and transcribes DNA for additional iron transport. It may be possible to hijack the machinery and engineer the receptor portion of the outer membrane (most of it is involved in transport, and we know which is which). Does not use dimerization, but rather uses conformational changes.
- OmpR - Naturally the osmolarity signal; used in the Texas project last year, but the actual screening was done at UCSF, and might not be a useful tactic for us.
- TonB - spans the periplasm! We just found it however, and don't know that much about it.
- Potential Solutions:
- Traverse the periplasm.
- Linker that attaches ToxR through the periplasm to an OMP that has antibodies attached.
- Find a protein that traverses the periplasm, and fuse the cytoplasmic domain to our signal cascade of choice (ToxR), and fuse the extracellular domain to antibodies.
- Directly fuse antibodies to outermembranen proteins that dimerize and initiate a signalling cascade -- would be ideal but we haven't found a system that does this.
- Express proteins where they do not belong. For example, try to tag the ToxR such that it will be expressed on the outer membrane.
- Just accept the fact that our system can only recognize small antigens, of sizes on the order of an amino acid or smaller, and go from there. This way, we can rely on diffusion through the outer membrane and cell wall.
MidWk Mtg 6/29 NOTES
- FK506 (?)
- FK1012 (dimer of FK506)
- Anti-"easily avail. chem"
- Easy to obtain
- Has lots of sequences
- Not prim/sec structure
Testing for Presence
- Fluorescence beads
- Fluor. Tag
- Rabbits have a lot.
- Affinity Chromatography - LAST RESORT
- Protein reside in periplasm
- Shuttle outer/inner
- Used for energy transduction
- Problem: autodimerizes?
- periplasm - 10 nm (100 atoms)
- FecA - signal is actually transduced!
People to See
- Graham Walker - 68-633
- Tim Clackson - Ariad Pharm. in Boston.
- Daniel Kahne - outermembrane protein fusion in E. coli . firstname.lastname@example.org
- UCSF Voigtlab - Ask about how to select the library (http://www.voigtlab.ucsf.edu/) Anselm Levskaya