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Team:CU-Boulder/Project/Kit/Purification
From 2013.igem.org
- ELPs - Elastin-like Proteins
- Elastin-like proteins are simply oligomeric repeats of Val-Pro-Gly-Xaa-Gly (Xaa being any amino acid with the exception of proline). ELPs undergo reversible, inverse phase transitions at at a transition temperature or after the addition of NaCl. Below this temperature/concentration of NaCl, ELPs are soluble. So here at CU-Boulder, we are trying to take advantage of this simple, yet interesting trait of these proteins by attempting to attach these ELPs to proteins in an attempt to come up with an easy and affordable method of protein purification. This method has been proven to work in previous experiments, so we're trying to develop a iGEM part that will include an optimized ELP that will make protein purification a simple process.
- RTX - Repeats in Toxin
- RTX is a structural motif consisting of a repeating set of amino acids that allows for precipitation in the presence of calcium. The RTX protein is intrinsically disordered under physiological conditions but undergoes a conformation change upon binding to calcium, otherwise known a ligand-induced disorder-to-order transition, which results in precipitation from solution. Our goal was to take advantage of this characteristic for the purpose of purifying proteins.
- Gel Purification using Color Tags
- We have developed a method of protein purification using color tags that allows us to separate proteins on a standard agarose gel.
- Detailed Protocol
- Yield and Purity Calculations
First, in this time lapse video, AmilCP and dsRED are separated on a .5% agarose gel for 120min
To improve yield, a barrier of standard computer paper is inserted into the path of the protein. This causes the protein to concentrate at the barrier.
Finally, the concentrated protein is cut, frozen, and spun in a homemade minicolumn to extract pure DNA free protein in TAE from the agarose matrix.
