Difference between revisions of "Team:UCLA/Notebook/Protein Cages"

Met with Sri and Todd to give feedback on our site

Main points listed below (paraphrased from Sri's notes on Slack):

• Site 1/4 looked pretty good; but the major thing is to do insertions rather than replacements on the protein
• Try to have least one G on each side of insertion
• so for site 1 (300-301), after S it would be SGLVPRGSG
• Todd will ask in the lab that there might be a few variants that might be useful (nicer looking cages, easier to purify, etc.)
• Will likely do a combination of gene synthesis (~4 constructs) and site-directed mutagenesis (to allow for more sites and linker variations)
• Will attempt about 12 constructs

Other ideas to think about:

• Using cysteines for chemical modifications (can use for some interesting assays to test cage disassociation)
• Could encapsulate a protease inside the cage to trigger a protease cascade (might be tough with current design)
• Would be interesting to see if we can replace all cysteines with serine or alanine

Found ~8 potential sites using PyMol and DSSP figures for 3vdx 16nm 12-subunit cage

Thrombin: Leu-Val-Pro-Arg-Gly-Ser (LVPRGS)

Linker: 264-294 (by inspection)

Potential Mutation Sites

• 297-302 (IIPSGP → LVPRGS) *least aa change + near linker
• 31-36 (GFPLSG → LVPRGS)
• 125-130 (LEPFLL → LVPRGS)
• 134-139 (DNPDGA → LVPRGS) *highly accessible to protease
• 217-222 (DVPALI → LVPRGS)
• 230-235 (TLPIEN → LVPRGS)
• 370 - 375 (GDPNNM → LVPRGS)
• 331-336(TRPILS→ LVPRGS)