One of the major aspects of our system utilizes metal binding proteins, so it was imperative that we purify the protein in order to move forward and complete our Copper Binding Assay. Our team successfully purified the Cut A protein through Nickel Affinity Chromatography (link) and Size Exclusion Chromatography (link).
The CutA protein was expressed in BL21 E.coli cells, and those cells were lysed then centrifuged to separate the supernatant and cell pellet. The lysate was then introduced to a Nickel Sepharose affinity column to isolate the CutA protein as it was bound to the Nickel Sepharose by its histidine tag. Then after washing to remove the unwanted proteins and cell debris, the CutA protein was eluted from the Nickel Sepharose.
Figure 1 - 15% SDS-PAGE of Nickel Affinity batch purification of CutA.
In the first lane we used the molecular weight marker RMR002 from GMbiolab. Lanes 2-5 show the elutions that contain our CutA protein. CutA protein runs at around 12kDA; however, in our SDS-PAGE gel it is seen at around 14kDa, this is likely because of the histidine tag. The remaining lanes are as follows: 6- Nickel Regeneration; 7- Cell Lysate Before Binding; 8- Cell Lysate After Binding; 9- Wash Sample; 10- Cell Pellet.
Figure 2 - 15% SDS-PAGE of Size Exclusion Chromatography elution samples.The first column contains the molecular weight marker RMR002 from GMbiolab, and lanes 2-16 contain our elutions. To further purify CutA, we ran the partially purified protein solution through a column, that contained beads. The beads have small crevices and this causes proteins of different sizes to pass through during different times.
(Link to protocol)
Figure 3 - Chromatograph demonstrating the peak of CutA protein. This figure is a chromatograph of the Size Exclusion Chromatography purification of the CutA protein. The A280 absorbance was read over time as the sample was eluted off the column. The resulting peak shows the elution volumes containing the CutA protein.
COPPER BINDING ASSAY
To begin, our team determined the standard copper concentration curve by measuring the average absorbance of various copper concentration solutions, and determined that the standard curve is linear. We can then relate the absorbance to the amount of copper left in solution; therefore, we can determine how efficient the metal binding proteins are, how many ions can be removed, the protein activity, optimal concentration of binding and optimal time of binding.
Figure 4 - Graph of standard copper concentration curve. Measured standard concentrations of copper in solution to provide a frame of reference for future copper binding assay.
Figure 5 - Graph demonstrating the absorbance of the copper solution after CutA (copper binding protein) was introduced as a function of time.Measured absorbance over various time intervals and determined that the optimal time is 60 minutes, and the optimal concentration is 151mg/L.
