Sandbox 323: Difference between revisions

The 3DS8 protein had already been sequenced, so comparisons between sequence and structure were carried out in a variety of programs to discern the possible function. After finding homologous and conserved regions from the databases, it was determined that the protein was an αβ-hydrolase. Once the function was proposed, common ligands were found to be modeled in active site docking software. During the molecular docking studies, PNPA was discovered to be a good ligand with multiple bonds to the active site of 3DS8. The protein vector was purchased and the 3DS8 protein was purified from the bacteria. The amount of protein in each column was quantified using a Bradford Assay to determine the elutions with the jighest concentration of protein. Some elutions had a negative amount, due to either being out of range of the plot or instrumental variation. Some of the samples were remade in order to correctly quantify them using the Bradford Assay. To verify the correct protein was purified from the column, gel electrophoresis was run. The gel confirmed a protein around 30 kDa, which was proposed earlier. To assess the activity of the protein, the protein was going to be assessed with the various ligands found in the molecular docking studies using UV-Vis. Many of the other possible substrates, such as PNP Butyrate, were on backorder but PNPA was delivered. The activity of the protein was all baseline, even after having complete saturation of the ligand in protein solution at various pH. After much confusion, it was concluded that the protein was no longer viable. Unfortunately, since the project was done over a couple of weeks, the concentration of protein in the elutions was too high and crashed out. The raw data about the 3DS8 activity was inconclusive.