Project Description

The human race moved from the stone of the Neolithic period and into the metallurgy of the Chalcolithic period with the widespread use of metal tools in their every day work regime (Gale 1991). From here little changed over the next 6,000 years where metals are still a massive part of our everyday lives but in quantities that dwarf previous usages. These high levels of metal requirements have left the landscape scarred.

Our project was inspired by the work done in the identification of platinum and palladium particles that are present on roads; emitted from catalytic converters (Deplanche, Murray et al. ). In 2010 50% of the worlds production of platinum and palladium were used for catalytic converters, with the highest amount being in Europe (Jollie 2010). Platinum and palladium appear ion the roads in small concentrations and in minute particles, < 3 μm (Prichard, Fisher 2012). These precious elements are a finite resource meaning that once its gone its gone. This doesn’t have to be the case, as the metals can be collected up and recycled.

The collection process is what we have been focussing on specifically. We have worked in a similar way to Chung et al, in that we have produced a protein with a metal binding peptide on the N-terminus of an easily expressible protein (Chan Chung, Cao et al. 2008). The difference is that we used glutathione S-transferase (GST tag) rather than ubiquitin, and instead of adding the binding peptide chemically to the protein we expressed both the protein and the peptide in E. Coli BL21 (DE3) cells. The peptides were taken from various sources (Seker, Demir 2011, Bae, Chen et al. 2000, Song, Caguiat et al. 2004, White, Liljestrand et al. 2007) and back translated specifically for E. coli using an online program Protein to DNA.

Protein

We decided to go down two different routes. The first was making an array of protein that bind a specific metal, and the second was to make a protein that has multiple metal binding sites.

With the first of the two ideas we thought that it would be possible to make a column, using the GST tag to bind the proteins in place. A solution containing multiple metal ions would then be passed through the column and bind each metal ion specifically. We initially looked at binding palladium, platinum and nickel (Seker, Demir 2011). The choice to use these metals was first, because platinum and palladium are the precious metals we were initially looking at, and second, because nickel columns were readily available to test this idea.Project Description

The second Idea is more of a scavenging protein. gBlocks (≤ 500 bps) were specifically designed and inserted into a pGEX-6P-1. One of which was designed to bind toxic metals such as cadmium, mercury and cobalt (Seker, Demir 2011, Bae, Chen et al. 2000, Song, Caguiat et al. 2004, White, Liljestrand et al. 2007). This was done by inserting flexible linkers between the metal binding sites (Hu, Wang et al. 2007). The linkers were used because they were quite flexible. This prevented hairpins in the structure. The second gBlock designed was for precious metals, not including platinum and palladium. The corresponding base pairs of gold, silver, aluminum and titanium peptides (Seker, Demir 2011) were used. The design of this differed from the other gBlock as myoglobin was hijacked, the loops removed and replaced with our metal binding peptides. .