Team:Cornell/project/background/metallothionein

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<h2 style="margin-top: 0px;">What are they?</h2>
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Metallothioneins are a group of low-molecular-weight proteins that rapidly bind with divalent metal ions.<sup>[1]</sup> Metallothioneins exist in almost every living organism and have many functions ranging from the control of oxidative stress to the regulation of redox potentials to the protection against toxic heavy metals.<sup>[1]</sup> For our project, we will take advantage of this final function where our recombinantly expressed metallothioneins will permanently sequester metals intracellularly while simultaneously providing resistance to engineered cells.  
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Far far away, behind the word mountains, far from the countries Vokalia and Consonantia, there live the blind texts. Separated they live in Bookmarksgrove right at the coast of the Semantics, a large language ocean. A small river named Duden flows by their place and supplies it with the necessary regelialia.
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Far far away, behind the word mountains, far from the countries Vokalia and Consonantia, there live the blind texts. Separated they live in Bookmarksgrove right at the coast of the Semantics, a large language ocean. A small river named Duden flows by their place and supplies it with the necessary regelialia.
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But I must explain to you how all this mistaken idea of denouncing pleasure and praising pain was born and I will give you a complete account of the system, and expound the actual teachings of the great explorer of the truth, the master-builder of human happiness.
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<h2>Mode of binding</h2>
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Metallothionein proteins across species have highly conserved sequences with approximately 30% of all residues as cysteines.<sup>[2]</sup> All cysteine residues in the metallothionein interact through a thiol group to coordinate a total of 7 divalent metal ions per protein.<sup>[3]</sup> These metal-thiolate bonds are quite strong, but what makes the binding of these metals to metallothionein so strong is that when bound the protein will change conformation to wrap almost completely around the coordinated metal ions.<sup>[3]</sup> For our project we will be taking advantage of this incredibly strong binding to sequester the toxic metals.
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The quick, brown fox jumps over a lazy dog. DJs flock by when MTV ax quiz prog. Junk MTV quiz graced by fox whelps. Bawds jog, flick quartz, vex nymphs. Waltz, bad nymph, for quick jigs vex! Fox nymphs grab quick-jived waltz. Brick quiz whangs jumpy veldt fox. Bright vixens jump; dozy fowl quack.
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No one rejects, dislikes, or avoids pleasure itself, because it is pleasure, but because those who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Nor again is there anyone who loves or pursues or desires to obtain pain of itself, because it is pain, but because occasionally circumstances occur in which toil and pain can procure him some great pleasure. To take a trivial example, which of us ever undertakes laborious physical exercise, except to obtain some advantage from it? But who has any right to find fault with a man who chooses to enjoy a pleasure that has no annoying consequences, or one who avoids a pain that produces no resultant pleasure? On the other hand, we denounce with righteous indignation and dislike men who are so beguiled and demoralized by the charms of pleasure of the moment, so blinded by desire, that they cannot foresee.
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Yeast Metallothionein with Cysteines
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Yeast Metallothionein Bound to Silver
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Yeast Metallothionein Surface Model
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<h1>Header 3</h1>
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<h2>GST-YMT</h2>
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The quick, brown fox jumps over a lazy dog. DJs flock by when MTV ax quiz prog. Junk MTV quiz graced by fox whelps. Bawds jog, flick quartz, ex nymphs. Waltz, bad nymph, for quick jigs vex! Fox nymphs grab quick-jived waltz. Brick quiz whangs jumpy veldt fox. Bright vixens jump; dozy fowl quack.
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The metallothionein gene we are utilizing with is <i>crs5</i>, which codes for YMT, or yeast metallothionein from <i>Saccharomyces cerevisiae</i>.<sup>[4]</sup>  Metallothioneins like YMT are fairly unstable as proteins and are degraded regularly in cells (on the timescale of hours), which would prevent a sequestration system from working effectively.<sup>[2]</sup>  To combat this issue we are working with YMT fused to glutathione-S-transferase from <i>Schistosoma japonicum</i> in a well established fusion protein system.<sup>[5]</sup>  This fusion should help stabilize the metallothionein and prevent action of lyases. This GST-YMT fusion system has been used previously and we will be utilizing it with a different regulatory system.<sup>[6]</sup>
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<h1>References</h1>
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<li>Ref 1</li>
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<li>Coyle, P., Philcox, J., Carey, L., & Rofe, A. (2002). Metallothionein: The multipurpose protein. <i>Cellular and Molecular Life Sciences</i>, 627-647.</li>
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<li>Ref 2</li>
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<li> Klaassen, C., Liu, J., & Choudhuri, S. (1999). METALLOTHIONEIN: An Intracellular Protein To Protect Against Cadmium Toxicity. <i>Annual Review of Pharmacology and Toxicology</i>,267-294.</li>
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<li>Ref 3</li>
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<li>Carpenè, E., Andreani, G., & Isani, G. (2007). Metallothionein functions and structural characteristics. <i>Journal of Trace Elements in Medicine and Biology</i>, 35-39.</li>
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<li>Culotta, V., Howard, W., & Liu, X. (1994). CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. <i>J. Biol. Chem.</i>, 269(41), 25295-25302.</li>
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<li>Smith, D., & Johnson, K. (1988). Single-step Purification Of Polypeptides Expressed In <i>Escherichia coli</i> As Fusions With Glutathione S-transferase. <i>Gene<i>, 31-40.</li>
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<li>Chen, S., & Wilson, D. (1997). Construction and characterization of <i>Escherichia coli</i> genetically engineered for bioremediation of Hg(2+)-contaminated environments. <i>Applied and Environmental Microbiology</i>, 63(6), 2442-2445.</li>
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Latest revision as of 03:29, 18 October 2014

Cornell iGEM

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Project Background

What are they?

Metallothioneins are a group of low-molecular-weight proteins that rapidly bind with divalent metal ions.[1] Metallothioneins exist in almost every living organism and have many functions ranging from the control of oxidative stress to the regulation of redox potentials to the protection against toxic heavy metals.[1] For our project, we will take advantage of this final function where our recombinantly expressed metallothioneins will permanently sequester metals intracellularly while simultaneously providing resistance to engineered cells.

Mode of binding

Metallothionein proteins across species have highly conserved sequences with approximately 30% of all residues as cysteines.[2] All cysteine residues in the metallothionein interact through a thiol group to coordinate a total of 7 divalent metal ions per protein.[3] These metal-thiolate bonds are quite strong, but what makes the binding of these metals to metallothionein so strong is that when bound the protein will change conformation to wrap almost completely around the coordinated metal ions.[3] For our project we will be taking advantage of this incredibly strong binding to sequester the toxic metals.
Yeast Metallothionein with Cysteines
Yeast Metallothionein Bound to Silver
Yeast Metallothionein Surface Model

GST-YMT

The metallothionein gene we are utilizing with is crs5, which codes for YMT, or yeast metallothionein from Saccharomyces cerevisiae.[4] Metallothioneins like YMT are fairly unstable as proteins and are degraded regularly in cells (on the timescale of hours), which would prevent a sequestration system from working effectively.[2] To combat this issue we are working with YMT fused to glutathione-S-transferase from Schistosoma japonicum in a well established fusion protein system.[5] This fusion should help stabilize the metallothionein and prevent action of lyases. This GST-YMT fusion system has been used previously and we will be utilizing it with a different regulatory system.[6]

References

  1. Coyle, P., Philcox, J., Carey, L., & Rofe, A. (2002). Metallothionein: The multipurpose protein. Cellular and Molecular Life Sciences, 627-647.
  2. Klaassen, C., Liu, J., & Choudhuri, S. (1999). METALLOTHIONEIN: An Intracellular Protein To Protect Against Cadmium Toxicity. Annual Review of Pharmacology and Toxicology,267-294.
  3. Carpenè, E., Andreani, G., & Isani, G. (2007). Metallothionein functions and structural characteristics. Journal of Trace Elements in Medicine and Biology, 35-39.
  4. Culotta, V., Howard, W., & Liu, X. (1994). CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J. Biol. Chem., 269(41), 25295-25302.
  5. Smith, D., & Johnson, K. (1988). Single-step Purification Of Polypeptides Expressed In Escherichia coli As Fusions With Glutathione S-transferase. Gene, 31-40.
  6. Chen, S., & Wilson, D. (1997). Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg(2+)-contaminated environments. Applied and Environmental Microbiology, 63(6), 2442-2445.

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