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An unclassified protein in the Protein Data Bank, 3H04, was selected as a protein of interest for kinetic and enzymatic analysis. The function of this unclassified protein, originating from the organism Staphylococcus aureus subsp. aureus Mu50, has not been previously identified. A thorough analysis was performed utilizing both wet lab and in silico techniques to gain key information on the function, enzymatic activity, and kinetic parameters of protein 3H04. This research was guided using protocols adapted from the Biochemistry Authentic Scientific Inquiry Lab (BASIL) Project. Structural information was gathered with in silico research, including bioinformatic databases such as BLAST and Pfam. The coding sequence for the unclassified protein was introduced into a vector and bacterial cells were transformed. To determine the function of 3H04, this protein was expressed and purified using affinity chromatography, and an SDS PAGE confirmed purification. A Bradford assay was used to determine the concentration of our protein, which was 0.579mg/mL. Lastly, a p-nitrophenol assay was used to determine enzymatic and kinetic activity of 3H04. Based on the data gathered, the unclassified protein is an enzyme with hydrolase activity.
Dyszel, Britney and Raffier, Nate, "Analysis on the Enzymatic Activity and Function of a Previously Unclassified Protein 3H04" (2021). Biochemistry and Molecular Biology Presentations. 18.
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