Expression and activity assay of recombinant flap endonuclease 1

Nan Sheng1 Yinjiao Ma1 Jianping Wang1 Bingjie Zou2 Guohua Zhou1,3

(1.School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu, China 210009)
(2.Department of Pharmacology, Nanjing General Hospital, Nanjing, Jiangsu, China 210002)
(3.Department of Pharmacology, Nanjing General Hospital, Nanjing, Jiangsu, China 210009)

【Abstract】Flap endonuclease 1 (FEN1) is an endonuclease that catalyzes invasive reaction. It can be used in signal-amplification reaction-based nucleic acid assay. However, the application of FEN1 is hampered due to the lack of detailed protocols to express and purify the enzyme, and to quantify the enzyme activity. In this paper, the DNA fragment coding the gene of FEN1 from Archaeoglobus fulgidus was synthesized, and inserted into the plasmid of pET24a(+) to express recombinant FEN1 with His-tag. After optimizing the expression, detailed expression protocol of FEN1 was obtained by culturing the recombinant E. coli at 37 °C with 200 r/min of shaking for 8 h, followed by inducing with 0.05 mmol/L IPTG at 37 °C for 11 h. The purified recombinant FEN1 with the molecular mass of 38 kDa was obtained by Ni-affinity chromatography. Moreover, we developed a accurate quantification method with fluorescence-labelled probes. Finally, the recombinant FEN1 was used in real-time PCR coupled with high specific invader assay for aldh2 gene genotyping to obtain the correct typing results, indicating that the recombinant FEN1 can be used in gene polymorphism detection. We provide a reliable enzyme for developing invasive reaction-based nucleic acid assay.

【Keywords】 flap endonuclease 1; signal amplification; activity assay;

【DOI】

【Funds】 National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2013ZX10004103) National Key Science&Technology Special Project(No.2013ZX10004103) Jiangsu Province's Clinical Science & Technology Special Project (No. BL2012061) Jiangsu Province’s Clinical Science&Technology Special Project(No.BL2012061) National Natural Science Foundation of China (Nos. 31300704, 21475151, 21405176, 21275161) National Natural Science Foundation of China(Nos.31300704,21475151,21405176,21275161) China Postdoctoral Science Special Foundation (Nos. 2013T60938, 2014T71011) China Postdoctoral Science Special Foundation(Nos.2013T60938,2014T71011) Six Talent Peaks Project in Jiangsu Province (No. 2015-WSN-085) Six Talent Peaks Project in Jiangsu Province(No.2015-WSN-085)

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(Translated by FANG Huaming)

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 10, Pages 1433-1442

October 2016

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Abstract

  • 1 Materials and methods
  • 2 Results and Analyses
  • 3 Discussion
  • References