EXPRESSION AND PURIFICATION OF PFU DNA POLYMERASE BELONGING TO THE B FAMILY POLYMERASE

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KHALILI BOROUJENI ZAHRA; ABEDI DARYOUSH; ABBASIAN MAHDI; MOFID MOHAMMAD REZA

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Paper Information

Journal: JOURNAL OF ISFAHAN MEDICAL SCHOOL (I.U.M.S) Year:2013 | Volume:31 | Issue:251 Page(s): 1392-1404

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Title

EXPRESSION AND PURIFICATION OF PFU DNA POLYMERASE BELONGING TO THE B FAMILY POLYMERASE

Author(s)

KHALILI BOROUJENI ZAHRA | ABEDI DARYOUSH | ABBASIAN MAHDI | MOFID MOHAMMAD REZA | Issue Writer Certificate

Keywords

DNAQ2

DEPENDENT DNA POLYMERASEQ3

POLYMERASE CHAIN REACTION (PCR)Q2

CLONINGQ2

PETQ2

15BQ2

Abstract

Background: DNA polymerases are enzymes directing the synthesis of DNA molecules from deoxyribonucleotides. They are essential tools for molecular biology. Pfu DNA polymerase was initially isolated from Pyrococcus furiosus, anaerobic hyperthermophilic archaeon lived in geothermally heated marine sediments with temperatures between 90oC and 100oC. This enzyme possesses 3’ to 5’ exonucleotic activity; so that makes correcting the errors made in DNA replication.Methods: In this research, the DNA fragment encoding Pfu DNA polymerase was cloned in to PET-15b expression vector. Then, by changing expression conditions such as isopropyl b-D-1-thiogalactopyranoside (IPTG) concentration, and time and temperature of the induction, the expression of this enzyme was optimized. Finally, Pfu DNA polymerase was produced in large scale in optimized conditions and purified with simple method. Then, purified enzyme was used in polymerase chain reaction (PCR) for evaluating the activity.Findings: Transformation of recombinant vector produced some colonies that most of them have the plasmid. The expression of Pfu DNA polymerase resulted in a bond approximately 90 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Maximum amount of protein production was observed in IPTG concentration of 0.75 mM, at 37oC, 3 hours incubation.Conclusion: Protein purification with using the method based on Desai protocol caused a product that had the activity like commercial one in PCR reaction.

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APA: Copy

KHALILI BOROUJENI, ZAHRA, ABEDI, DARYOUSH, ABBASIAN, MAHDI, & MOFID, MOHAMMAD REZA. (2013). EXPRESSION AND PURIFICATION OF PFU DNA POLYMERASE BELONGING TO THE B FAMILY POLYMERASE. JOURNAL OF ISFAHAN MEDICAL SCHOOL (I.U.M.S), 31(251), 1392-1404. SID. https://sid.ir/paper/50579/en

Vancouver: Copy

KHALILI BOROUJENI ZAHRA, ABEDI DARYOUSH, ABBASIAN MAHDI, MOFID MOHAMMAD REZA. EXPRESSION AND PURIFICATION OF PFU DNA POLYMERASE BELONGING TO THE B FAMILY POLYMERASE. JOURNAL OF ISFAHAN MEDICAL SCHOOL (I.U.M.S)[Internet]. 2013;31(251):1392-1404. Available from: https://sid.ir/paper/50579/en

IEEE: Copy

ZAHRA KHALILI BOROUJENI, DARYOUSH ABEDI, MAHDI ABBASIAN, and MOHAMMAD REZA MOFID, “EXPRESSION AND PURIFICATION OF PFU DNA POLYMERASE BELONGING TO THE B FAMILY POLYMERASE,” JOURNAL OF ISFAHAN MEDICAL SCHOOL (I.U.M.S), vol. 31, no. 251, pp. 1392–1404, 2013, [Online]. Available: https://sid.ir/paper/50579/en

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