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Revision as of 07:27, 16 October 2014

Results

Protein stability analysis using Circular Dichroism

Our system was verified using Circular Dichroism (CD) analysis. A scan of the protein in solution in PBS was scanned across a variety of wavelengths to find the signal minima that would best indicate the state of folding. An equivalent concentration of protein in concentrated guanidinium chloride (GdmCL), a powerful chaotropic agent, was then prepared to be mixed in to our sample. This solution was then added to the sample in small increments, allowing us to measure the CD signal at increasing concentrations of GdmCl while maintaining a constant concentration of the protein being tested. The concentration of GdmCl at which the CD signal was half of its initial value was recorded. A higher concentration of GdmCl being required to half denature a protein indicates greater stability.

Protein expression analysis using SDS-PAGE

Samples of our protein of interest were taken at various points during the purification cycle. These samples were then run through gel electrophoresis to determine the relative amounts of protein produced by the cells. Larger bands were indicative of greater expression.

Protein stability analysis using Degron Constructs and Flow Cytometry

The results for the no-protein of interest degron constructs matched expected results in which Deg0 had the highest expect stability which correlated to the highest fluorescence output. These expectations were validated by our experiment results in which cells with Deg0 exhibited the highest fluorescent levels. Our second expectations were that Deg2 and Deg3 would exhibit middle levels of fluorscence, lower than Deg0 but higher than Deg1 or deg4. Once again, these expectations were validated by our experimental results. The results for the no protein of interest degron constructs are as follow, cells containing our Deg0 protein construct exhibited the highest fluorescent followed by Deg2 and deg3 and finally by Deg1 and Deg4 both of which exhibited baseline levels (no protein, no degron construct PYE1 cells) of fluorescence.
In order, to validate the system as whole we must analyze the degron constructs with a specific well studied protein to analyze that protein's stability with our system compared to the protein stability measurements from current existing techniques. There were three variants of a protein each with varying stabilities that were quantified using circular dichroism and guanidium hydrogen chloride melts. The protein BINDI had the highest stability followed by BbpD04.3 and then BbpD04. If our system is accurate, cells containing the BINID-Degron construct would exhibit the highest fluorescence output followed by BbpD04.3 and BbpD04 would show the lowest fluorescence output. Since there are 5 possible degron construct for each of our 3 proteins of interest, all 15 data points would have to match our expectations. We expect that BINDI Deg0 would have the highest fluorescence of the all protein of interest Deg0 constructs followed by BbpD04.3 Deg0 and BbpD04 Deg0. Next, BINDI Deg2/3 would have middle levels of fluorescence followed by BbpD04.3 Deg2/3 and BbpD04 Deg2/3. Finally, BINDI Deg1/4, BbpD04.3 Deg1/4 and BbpD04 Deg1/4 would have the lowest levels of fluorescence if not baseline levels. The experimental results accquired through flow cytometry show a rough correlation to these expections********more experiment required***********

Mutagenesis Results and Mutant Variant Analysis