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

Revision as of 21:56, 17 September 2015

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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

We used Pymol software to model our protein cage and determine where to insert out thrombin cut site mutation.

Using the design restriction listed below, we narrowed down 10 possible locations for the insert

We successfully cloned and expressed one of our mutant cages and our wild type cage.

We ran dynamic light scattering on both but did not see correct size cage formation.

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

Cloning

Have not started yet. Will begin designing gBlocks and primers for site-directed mutagenesis early next week.

gBlocks designed and primers designed to amplify sequence and to add 6x His tag so that sequence can be cloned into vector with T7 and RBS.

PCR was done but we're getting nonspecific binding and are currently troubleshooting and optimizing.

Mutant 10 cage was successfully cloned.

Protein Expression

Long way to go till we get here.It's here!

We are expressing protein from the constructs that Yeates gave us.

We have expressed the wild type cage and the mutant 10 cage.

What we are working on now

We are currently narrowing and refining our list of selected mutation sites.

We are currently troubleshooting and optmimzing our PCR to optimize yield.

We look to get the yield we one and to start cloning into the T7/RBS igem vector.

We are currently protein expressing constructs that Yeates gave us and will be characterizing cage structure soon.

We are cloning our 9 other mutant cages and are planning to express them.

We are looking for cage formation at the appropriate size of our native and mutant 10 cages.

We will introduce thrombin and look for cleavage of our mutant protein cage structures.

Raw lab notebook entries