Optimization of the Production and Purification of Taq Polymerase Enzyme Containing N666E Mutation in its O-Helix Region

مجله دانشکده پزشکی اصفهان. 2012;30(193)

 

Journal Homepage

Journal Title: مجله دانشکده پزشکی اصفهان

ISSN: 1027-7595 (Print); 1735-854X (Online)

Publisher: Vesnu Publications

Society/Institution: Isfahan University of Medical Sciences

LCC Subject Category: Medicine: Medicine (General)

Country of publisher: Iran, Islamic Republic of

Language of fulltext: Persian

Full-text formats available: PDF, XML

 

AUTHORS

Hamid Mirmohammad Sadeghi (Professor, Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran)
Mohammed Rabbani (Professor, Department of Pharmacology, School of Pharmacy and Pharmaceutical Sciences Isfahan University of Medical Sciences, Isfahan, Iran)
Samaneh Assarzadeh (PharmD Candidate, School of Pharmacy and Pharmaceutical Sciences, And Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran)
Fatemeh Moazen (Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran)

EDITORIAL INFORMATION

Double blind peer review

Editorial Board

Instructions for authors

Time From Submission to Publication: 18 weeks

 

Abstract | Full Text

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. Keywords: Taq polymerase recombinant, Purification, Desai protocol, O-helix mutation