Team:UCLA/Notebook/Protein Cages

iGEM UCLA




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/0B7kb5ShZyyqVcU0xWlVYbUVfSW8/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 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 10* 243-248 ALPSAE ALPGLVPRGSGSAE Satisfies 1, possibly 2 also, very exposed for thrombin cleavage
Site 13* 351-354 VPSE GLVPRGSGE Satisfies 1; only 4 insertions needed.
Site 6 228-233 DRTLPI DRTLGLVPRGSGPI 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 11 123-129 ASLEPFL ASLVPRGSGEPGL Sterically hindered. 6 insertions needed.
Site 12 318-321 KNTD KNLVPRGSGTD Satisfies 1, 7 insertions needed.
Site 13 190-196 AASGGFF AASGLVPRGSGFF Satisfies neither 1 nor 2, sterically hindered and close to secondary structures. Probably not good.

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

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