Optimization of the Production and Purification of Taq Polymerase Enzyme Containing N666e Mutation in Its O-Helix Region



Sadeghi HM^{1, 4} ; Rabbani M² ; Assarzadeh S³ ; Moazen F^{1, 4} Show Affiliations
Source: Journal of Isfahan Medical School Published:2012

Abstract

Background: The aim of this project was to isolate and purify the highly active recombinant Taq DNA polymerase from the strain of Escherichia coli BL21. This enzyme, with a molecular weight of about 94 kDa, is widely used in polymerase chain reaction (PCR). In PCR, the activity and fidelity of Taq polymerase can significantly influence the results. One of the active regions of Taq polymerase has been suggested to be the O-helix region. In previous studies, an expression vector containing mutated Asn 666 Glu Taq polymerase gene was designed. In order to investigate the effects of this mutation on the function of the enzyme, Taq polymerase needs to be purified first. Methods: In this study, after transformation of competent cells, enzyme expression was induced by isopropyl β D thiogalactopyranoside (IPTG) method. Modified Desai protocol with DNase and RNase, nickel-nitrilotriacetic acid (Ni-NTA) resin, trichloroacetic acid (TCA), and refolding protocols were subsequently used for purification of this enzyme. These protocols were finally compared. Findings: Using Desai protocol resulted in the production of a sharp band in the expected region (94 kDa) and several other visible bands. After further modification of Desai protocol, only the desirable band was observed. In protocols using TCA and Ni-NTA resin, the expected bands were weak. Refolding protocol caused a band in an undesirable region (66 kDa). Conclusion: From the different purification techniques that were used in this study, the modified method of Desai containing RNase and DNase worked best. Addition of TCA can precipitate proteins that had not been affected by heat. Using Ni-NTA resin resulted in elimination of unwanted bands. However, the amount of Taq polymerase was also decreased. The extra band that was observed in refolding protocol was probably due to the presence of proteases that were isolated with inclusion body and could digest Taq polymerase.