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Determining the role of a protein can be beneficial in understanding how certain mechanisms work or shed light on a new area of research. The purpose of this study is to determine the function of the protein with a PBD ID of 4DIU. An expression vector containing genes for E. Coli metabolism, ampicillin resistance, and the protein 4DIU were introduced to competent bacteria. These bacteria were then forced to express the protein using an Autoinduction Media TB. The expressed protein was collected and purified to be used in later analysis. Furthermore, an SDS-PAGE confirmed successful protein purification by showing a solid band around the correct weight of 28.45 kDa for protein 4DIU. A final Bradford assay determined the overall concentration of our protein to be 3.56 mg/mL. Subsequently, computational methods including Blast and Pfam provided background knowledge of protein 4DIU. It was found to be under the abhydrolase superfamily and contained hydrolase_4 and serine aminopeptidase S33 domains. We hypothesize that its function most likely falls under the role of a carboxylesterase or lipase. This was tested by performing an enzyme kinetics assay using PNAP as a substrate to test the function of a hydrolase.
Merlo, Kyle and McGinley, Kaitlyn, "Identification and Analysis of the Function for Protein 4DIU" (2021). Biochemistry and Molecular Biology Presentations. 15.
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