Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli

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• 作者：Wenjun Zheng ; Qingsong Wang ; Qun Bi
• 关键词：Pfu DNA polymerase ; Polymerase chain reaction ; Peptide mass fingerprint ; Isoelectric point
• 刊名：The Protein Journal
• 出版年：2016
• 出版时间：April 2016
• 年：2016
• 卷：35
• 期：2
• 页码：145-153
• 全文大小：1,387 KB
• 参考文献：1.Lundberg KS, Shoemaker DD, Adams MWW, Short JM, Sorge JA, Mathur EJ (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1–6CrossRef
  2.Ppyun H, Kim I, Cho SS, Seo KJ, Yoon K, Kwon S (2012) Improved PCR performance using mutant Tpa-S DNA polymerases from the hyperthermophilic archaeon Thermococcus pacificus. J Biotechnol 164:363–370CrossRef
  3.Cline J, Braman JC, Hogrefe HH (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 24:3546–3551CrossRef
  4.Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004) Recent developments in the optimization of thermostable DNA polymerases for efficient applications. Trends in Biotechnol 22:253–260CrossRef
  5.Kim YJ, Cha S, Lee HS, Ryu YG, Bae SS, Cho Y, Cho H, Kim S, Kwon S, Lee J, Kang SG (2008) Sensing domain and extension rate of a family B-Type DNA Polymerase determine the stalling at a deaminated base. J Microbiol Biotechnol 18:1377–1385
  6.Norholm MHH (2010) A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol 10:21–27CrossRef
  7.Jozwiakowski SK, Connolly BA (2009) Plasmid-based lacZa assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase. Nucleic Acids Res 37:102–108CrossRef
  8.Melissis S, Labrou NE, Clonis YD (2007) One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography. J Biotechnol 2:121–132CrossRef
  9.Lu C, Erickson HP (1997) Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus. Protein Expr Purif 11:179–184CrossRef
  10.Hu J, Wang F, Liu C (2015) Development of an efficient process intensification strategy for enhancing Pfu DNA polymerase production in recombinant Escherichia coli. Bioprocess Biosyst Eng 38:651–659CrossRef
  11.Ferralli P, Egan J (2007) Making Taq DNA polymerase in the undergraduate biology laboratory. Bios 78:69–74CrossRef
  12.Sun ZH, Cai J (2006) Purification of recombinant Pfu DNA polymerase using a new JK110 resin. Korean J Chem Eng 23:607–609CrossRef
  13.Nika H, Angeletti RH, Hawke DH (2014) N-terminal protein characterization by mass spectrometry using combined microscale liquid and solid-phase derivatization. J Biomol Tech 25:77–86CrossRef
  14.Uemori T, Ishino Y, Toh H, Asada K, Kato I (1993) Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. Nucl Acids Res 21(2):259–265CrossRef
  15.Ignatov KB, Barsova EV, Fradkov AF, Blagodatskikh KA, Kramarova TV, Kramarov VM (2014) A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification. Biotechniques 57:81–87
  16.Braithwaite DK, Ito J (1993) Compilation, alignment, and phylogenetic relationships of DNA polymerases. Nucleic Acids Res 21(4):787–802CrossRef
  17.Kim SW, Kim DU, Kim JK, Kang LW, Cho HS (2008) Crystal structure of Pfu, the high fidelity DNA polymerase from Pyrococcus furiosus. Int J Biol Macromol 42:356–361CrossRef
  18.Drum M, Kranaster R, Ewald C, Blasczyk R, Marx A (2014) Variants of a Thermus aquaticus DNA polymerase with increased selectivity for applications in allele- and methylation-specific amplification. PLoS ONE 9(5):e96640–e96650CrossRef
  19.Biles BD, Connolly BA (2004) Low-fidelity Pyrococcus furiosus DNA polymerase mutants useful in error-prone PCR. Nucleic Acids Res 32(22):176–182CrossRef
  20.Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, Imanaka T (1997) Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl Environ Microbiol 63(11):4504–4510
  21.Kim YJ, Lee HS, Bae SS, Jeon JH, Lim JK, Cho Y, Nam KH, Kang SG, Kim SJ, Kwon ST, Lee JH (2007) Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1. J Microbiol Biotechnol 17(7):1090–1097
  22.Mroczkowski BS, Huvar A, Lernhardt W, Misono K, Nielson K, Scott B (1994) Secretion of thermostable DNA polymerase using a novel baculovirus vector. J Biol Chem 269(18):13522–13528
  23.Miura M, Tanigawa C, Fujii Y, Kaneko S (2013) Comparison of six commercially-available DNA polymerase for direct PCR. Rev Inst Med Trop Sao Paulo 55(6):401–406CrossRef
  24.Viennois E, Chen F, Laroui H, Baker MT, Merlin D (2013) Dextran sodium sulfate inhibits the activities of both polymerase and reverse transcriptase: lithium chloride purification, a rapid and efficient technique to purify RNA. BMC Res Notes 6:360–367CrossRef
  25.Chong SS, Eichler EE, Nelson DL, Hughes MR (1994) Robust amplification and ethidium-visible detection of the fragile X syndrome CGG repeat using Pfu polymerase. Am J Med Genet 51(4):522–526CrossRef
  26.Eckert KA, Kunkel TA (1991) DNA polymerase fidelity and the polymerase chain reaction. Genome Res 1:17–24CrossRef
  27.Moser IJ, DiFrancesco RA, Gowda K, Klingele AJ, Sugar DR, Stocki S, Mead DA, Schoenfeld TW (2012) Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme. PLoS ONE 7(6):e38371–e38383CrossRef
  28.Barnes DE, Lindahl T (2004) Repair and genetic consequences of endogenous DNA base damage in mammalian cells. Annu Rev Genet 38:445–476CrossRef
  29.Krokan HE, Drablos F, Slupphaug G (2002) Uracil in DNA—occurrence, consequences and repair. Oncogene 21(58):8935–8948CrossRef
  30.Melissis S, Labrou NE, Clonis YD (2006) Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes one-step purification of Pfu DNA polymerase. J Chromatogr A 1122(1–2):63–75CrossRef
  31.Pearl LH (2000) Structure and function in the uracil-DNA glycosylase superfamily. Mutat Res/DNA Repair 460(3–4):165–181CrossRef
  32.Wardle J, Burgers PMJ, Cann IKO, Darley K, Heslop P, Johansson E, Lin LJ, McGlynn P, Sanvoisin J, Stith CM, Connolly BA (2008) Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya. Nucleic Acids Res 36(3):705–711CrossRef
  33.Dabrowski S, Ahring BK (2000) Cloning, expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR. Protein Expr Purif 19(1):107–112CrossRef
  34.Niimi H, Mori M, Tabata H, Minami H, Ueno T, Hayashi S, Kitajima I (2011) A novel eukaryote-made thermostable DNA polymerase which is free from bacterial DNA contamination. J Clin Microbiol 49(9):3316–3320CrossRef
  35.Kuroita T, Matsumura H, Yokota N, Kitabayashi M, Hashimoto H, Inoue T, Imanaka T, Kai Y (2005) Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases. J Mol Biol 351(2):291–298CrossRef
  36.Spangler R, Goddard NL, Thaler DS (2009) Optimizing Taq polymerase concentration for improved signal-to-noise in the broad range detection of low abundance bacteria. PLoS ONE 4(9):e7010–e7017CrossRef
  
• 作者单位：Wenjun Zheng (1)
  Qingsong Wang (1)
  Qun Bi (1)
  
  1. College of Life Sciences, Peking University, Beijing, 100871, People’s Republic of China
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Bioorganic Chemistry
  Biochemistry
  Organic Chemistry
  Animal Anatomy, Morphology and Histology
  
• 出版者：Springer Netherlands
• ISSN：1573-4943

文摘

Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His₆-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni2+ chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.