Team:Cornell/project/background/metallothionein

From 2014.igem.org

Cornell iGEM

web stats

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.

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.