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Our research is part of the Biochemistry Authentic Scientific Inquiry Lab (BASIL) project to use wet-lab and in-silico techniques to purify and characterize the function of unknown protein 3H04. Based on in-silico data protein 3H04 is predicted to be an alpha/beta hydrolase, specifically a serine protease. To test this hypothesis using wet-lab techniques, an expression vector containing the sequence of the protein and an antibiotic resistance gene was transformed into chemically competent E.coli. Protein expression was induced by utilizing the lac operon. His-tagged purification via affinity chromatography was performed. SDS-PAGE confirmed that 3H04 was successfully purified and has a molecular weight of about 73.9 kDa. Data from a Bradford assay indicated that the purified protein had a concentration of 2.40 mg/mL. An enzyme activity assay showed that 3H04 has an activity of 1.80x10 -3 µmol/min when the general hydrolase substrate p-nitrophenyl acetate (PNPA) was used. Kinetic analysis also showed 3H04 followed Michaelis Menten kinetics when PNPA was used as the substrate. The results in this study confirm that 3H04 is a hydrolase, and in the future, kinetic analysis will be conducted using a substrate that is specific for serine proteases in order to determine the specific function of 3H04
Chell, Stephanie and Ruiz, Kayla, "Characterization of Unknown Protein 3H04" (2021). Biochemistry and Molecular Biology Presentations. 11.
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