Paper- Restricted to Campus Access
The objective of this experiment was to purify and characterize the function of an unknown enzyme through both wet and computational lab protocols. Protein 3H04 was isolated and additional research was conducted using BLAST, PFAM, and DNASU to determine the characteristics and mechanism of this protein. We introduced an expression vector into E. coli via transformation in order to express the protein of interest (POI). Bacteria that took up the expression vector were selected under antibiotic pressure on plates, cultures were inoculated, and then transferred into autoinduction media in order to induce protein expression. The POI was isolated via affinity chromatography, confirmed by SDS PAGE, and the concentration was measured with a Bradford Assay. We successfully isolated 3H04 from E. coli at a concentration of 1.525 mg/mL. Para-nitrophenyl acetate and kinetic assays were also carried out in order to confirm our protein is a hydrolase and determine the Km, Vmax, and Km/Vmax values.
Paulson, Kayla and Wun, Jesse, "Isolation and Characterization of Protein 3H04" (2021). Biochemistry and Molecular Biology Presentations. 20.
Available to Ursinus community only.