Lal, ShaileshSadik, Tomas2022-06-232022-06-23http://hdl.handle.net/10323/11916Protein purification is an important biotechnological process used to induce the expression of a desired protein from host cells, isolate the protein using one or more established methods, and collect it in its purified form. Applications of such exist in pharmaceutical products, clinical research, and other medicinal areas. This process describes the execution and optimization of the protein purification of Taq polymerase, a DNA polymerase enzyme natively expressed by the bacterial species Thermus aquaticus. Introduction of the gene into the Escherichia coli host cells was completed using recombinant DNA technology, and expression of the desired Taq polymerase protein was induced via isopropyl-β-D-1-thiogalactopyranoside (IPTG). Its isolation and collection were facilitated via cell lysing and ion-exchange column chromatography. The resulting Taq polymerase was successfully utilized as DNA polymerase in the polymerase chain reaction. Optimization of the process was achieved by maximizing protein yield and consolidating fraction collection into the smallest possible number of fractions. This occurred in the form of two parameter modifications: IPTG inducing concentration and chromatography flow rate. Five concentrations of IPTG were tested based on the standard protocol value of 0.335 mM, those being 0.08375 mM, 0.1675 mM, 0.335 mM, 0.5025 mM, and 0.67 mM. Three flow rates were tested using peristaltic pump settings, those being 0.5 mL/min, 1 mL/min, and 2.0 mL/min. It was found that an IPTG concentration of 0.1675 mM yielded the highest protein expression, and the flow rate of 2.0 mL/min resulted in the highest Taq polymerase expression concentrated in a single fraction. These results hold value for resource and time savings of large-scale protein purification processes, and can be replicated and applied to the purification of therapeutic proteins, clinical protein study, and other fields.Taq polymeraseProtein purificationColumn chromatographyIon exchange column chromatographyRecombinant DNADNA polymerasePolymerase chain reactionThesis