Difference between revisions of "Team:UCLA/Notebook/Protein Cages"
(→Design) |
(→Design) |
||
| Line 89: | Line 89: | ||
| AASGGFF | | AASGGFF | ||
| AASG'''LVPRGS'''GFF | | AASG'''LVPRGS'''GFF | ||
| − | | Satisfies neither 1 nor 2, sterically hindered and close to secondary structures | + | | Satisfies neither 1 nor 2, sterically hindered and close to secondary structures. Probably not good. |
|- | |- | ||
| Site 4* | | Site 4* | ||
Revision as of 00:47, 27 May 2015
Contents
Goals
The goal of this project is to produce various mutants of a 3-dimensional protein fusion capable of self-assembling into a tetrahedral cage structure (PDB: 3VDX). These variants will have thrombin protease sites (LVPRGS) introduced in selected locations, allowing for dissociation of the cage structure upon thrombin treatment. Ultimately, we aim to develop a controllable system allowing for both drug-loading and release using the protein cage scaffold.
Achievements
As of 5/22, we have a detailed list of ~15 unique potential sites to insert into the protein cage. We will narrow this list to ~12 sites, and begin designing constructs beginning of next week. Four of our best sites will be synthesized through IDT, while the remaining will be produced through site directed mutagenesis.
Design
Following is a list of our insertion sites. Sites marked with asterisks are preferred sites. Sites were selected by examining the DSSP secondary structure to ensure minimal disruption of existing alpha-helices or beta sheets, ensuring sites were sandwiched by glycine residues, and using PyMOL to check if the site would be accessible to the protease. Top candidates are based on two main criteria:
1. Geometric accessibility by protease
2. Speculated extent of conformational changes by insertion (minimum preferred)
PyMOL PSE file with sites highlighted: https://drive.google.com/file/d/0B7kb5ShZyyqVb2tjSzd3SDZuc1k/view?usp=sharing
| Site Number | Residues | Original Sequence | Mutant Sequence | Notes |
|---|---|---|---|---|
| Site 1* | 298-305 | IPSGPLKA | IPSGLVPRGSGPLKA | Satisfies 1 and 2, depending on oligomerization geometry |
| Site 2* | 134-140 | DNPDGAA | DNPDGLVPRGSGAA | Satisfies 1 and 2 |
| Site 3 | 190-196 | AASGGFF | AASGLVPRGSGFF | Satisfies neither 1 nor 2, sterically hindered and close to secondary structures. Probably not good. |
| Site 4* | 252-258 | VEGAPHG | VEGLVPRGSGAPHG | Satisfies 1 and 2; is close to linker region |
| Site 5* | 331-336 | TRPILSP | TRPGLVPRGSGILSP | Satisfies 1 and 2, depending on oligomerization geometry |
| Site 6 | 228-233 | DRTLPI | DRTLGLVPRGSGIPI | Satisfies 1 |
| Site 7 | 216-220 | IDVPA | IDVGLVPRGSGPA | Satisfies 1, possibly 2, easily accessible and within longer turn |
| Site 8 | 64-72 | SSQPTTGYD | SSQPTTGLVPRGSGYD | Satisfies 1, but not ideally oriented for access by thrombin |
| Site 9 | 417-422 | RMGAVT | RMGLVPRGSGAVT | Satisfies 1, depending on geometry, possible steric hindrance (inner portion of cage) |
| Site 10* | 243-248 | ALPSAE | ALPGLVPRGSGSAE | Satisfies 1, possibly 2 also, very exposed for thrombin cleavage |
| Site 11 | 123-129 | ASLEPFL | ASLVPRGSGEPGL | Sterically hindered. 6 insertions needed. |
| Site 12 | 318-321 | KNTD | KNLVPRGSGTD | Satisfies 1, 7 insertions needed. |
| Site 13* | 351-354 | VPSE | LVPRGSGE | Satisfies 1; only 4 insertions needed. |
Cloning
Have not started yet. Will begin designing gBlocks and primers for site-directed mutagenesis early next week.
Protein Expression
Long way to go till we get here.
What we are working on now
We are currently narrowing and refining our list of selected mutation sites.
Raw lab notebook entries
| May | ||||||
| M | T | W | T | F | S | S |
| 1 | 2 | 3 | ||||
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| June | ||||||
| M | T | W | T | F | S | S |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| 22 | 23 | 24 | 25 | 26 | 27 | 28 |
| 29 | 30 | |||||
| July | ||||||
| M | T | W | T | F | S | S |
| 1 | 2 | 3 | 4 | 5 | ||
| 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 13 | 14 | 15 | 16 | 17 | 18 | 19 |
| 20 | 21 | 22 | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 | 31 | ||
| August | ||||||
| M | T | W | T | F | S | S |
| 1 | 2 | |||||
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| 31 | ||||||
| September | ||||||
| M | T | W | T | F | S | S |
| 1 | 2 | 3 | 4 | 5 | 6 | |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | ||||