Protein Expression and Purification of Taq Polymerase

Abstract

The development and application of molecular diagnostic technologies has triggered a revolution in infectious disease diagnosis and surveillance over the years. The ability to deliver a quick, specific, reliable, cost-effective, and straightforward alternative is one of the advantages of using molecular diagnostics. Taq polymerase is an example of an enzyme that is required in molecular diagnostics testing. Taq polymerase is a heat-resistant DNA polymerase derived from the extremophilic bacteria Thermus aquaticus. The purpose of this research is to confirm the presence of the ""Taq polymerase"" product generated from Escherichia coli BL21(DE3) pLysS bacteria and purify it. The experiment began with the growth of Escherichia coli BL21(DE3) pLysS bacteria, which had already been engineered to incorporate the Taq polymerase DNA fragment and linked to the DNA sequence that encodes the His-tag within the bacteria plasmid. The bacterium sample was in the form of a glycerol stock, and the bacterial growth was measured using the OD 600. The culture was then treated with 1 mM IPTG to induce the expression of the Taq polymerase protein. The protein produced as Taq polymerase was validated using SDS-PAGE and Western blot techniques. The method was then carried on to the purification phase, which involved the use of IMAC with Ni-NTA resin. The His-tag is specially bound to the resin and can assist in the separation of the Taq polymerase protein from other impurities. The purity percentage of the purified Taq polymerase protein was then determined using the ImageJ software. Lastly, the buffer after purification was changed into a storage buffer using the dialysis process, and the final concentration of the purified Taq polymerase was evaluated using absorbance at 280 nm. The final outcome showed that the Taq polymerase protein was successfully produced with a high purity percentage of 89%. However, the final protein quantification analysis after dialysis revealed only a 0.014 mg total protein concentration. As for the recommendation, it is recommended to continue the study to the activity evaluation step. Another purification step also can be performed to increase the purity percentage of the purified protein. Analyze the growth curve of bacteria, try experiments with different duration and concentration of IPTG induction also can be done in the future to maximize the protein concentration.