Team:CU-Boulder/Project/Kit/Purification

From 2013.igem.org

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<h3>ELPs - Elastin-like Proteins</h3>
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<dt>ELPs - Elastin-like Proteins</dt>
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<dd>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.</dd>
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<p>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.</p>
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We designed our ELP to be 100 amino acids long with 10% of the guest residues being tyrosine and 90% being valine. We designed the ELP to solubilize at around 40 degrees Celsius and an NaCl concentration of about .2M. We constructed the ELP because we wanted to create the easiest conditions to induce solubilization. If our method proves successful, then the only required materials to purify an ELP tagged protein would be a heat block and NaCl.
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1188004"> ELP Part </a>
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<h3>RTX - Repeats in Toxin</h3>
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<dt>RTX - Repeats in Toxin</dt>
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<dd>RTX is a motif consisting of a repeating set of amino acids that allows for precipitation in the presence of calcium...</dd>
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<p>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.</dd>
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<img src="https://static.igem.org/mediawiki/parts/5/5f/RTXPrecipitation.PNG" width = "500">
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<img src="https://static.igem.org/mediawiki/parts/e/eb/RTXSDSPage.PNG" width = "400">
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1188002">RTX tag Characterization</a></li>
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<dt>Gel Purification using Color Tags</dt>
<dt>Gel Purification using Color Tags</dt>
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<p></p>
<dd>We have developed a method of protein purification using color tags that allows us to separate proteins on a standard agarose gel.</dd>
<dd>We have developed a method of protein purification using color tags that allows us to separate proteins on a standard agarose gel.</dd>
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First, in this time lapse video, AmilCP and dsRED are separated on a .5% agarose gel for 120min
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<video width="640" height="360" controls>
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<source src="https://static.igem.org/mediawiki/2013/c/c6/TimeLapseRFPAmilCPSep.mp4" type="video/mp4">
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<a href="https://static.igem.org/mediawiki/2013/c/c6/TimeLapseRFPAmilCPSep.mp4">GoPro Protocol: Agarose Protein Separation</a>
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</video>
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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.
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<video width="640" height="360" controls>
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<source src="https://static.igem.org/mediawiki/2013/3/33/PaperBarrier.mp4" type="video/mp4">
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</video>
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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.
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<video width="640" height="360" controls>
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<source src="https://static.igem.org/mediawiki/2013/3/34/CutFreezeSpin.mp4" type="video/mp4">
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</video>
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<li><a href="https://2013.igem.org/Team:CU-Boulder/Notebook/Protocols/ProteinPurificationAgaroseGel">Detailed Protocol</a></li>
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<li><a href="https://2013.igem.org/Team:CU-Boulder/Notebook/Experiments/Experiment_14_1">Yield and Purity Calculations</a</li>
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Latest revision as of 15:37, 28 October 2013

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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.

We designed our ELP to be 100 amino acids long with 10% of the guest residues being tyrosine and 90% being valine. We designed the ELP to solubilize at around 40 degrees Celsius and an NaCl concentration of about .2M. We constructed the ELP because we wanted to create the easiest conditions to induce solubilization. If our method proves successful, then the only required materials to purify an ELP tagged protein would be a heat block and NaCl.

ELP Part

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.

    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.

  • Detailed Protocol
  • Yield and Purity Calculations

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