Expression, purification and characterization of flap endonuclease 1 from Pyrococcus furious

Abstract:

[Objective] To clone, express, purify and characterize flap endonuclease 1 from thermophilic archaea Pyrococcus furious.[Methods] We cloned fen1 gene from Pyrococcus furious (pFEN1), expressed it in Escherichia coli and purified the protein by affinity chromatography. We applied denaturing polyacrylamide gel electrophoresis to detect the enzymatic activities and studied its interaction with other proteins by using fluorescence labeled oligonucleotides as substrates.[Results] pFEN1 was overexpressed in E. coli. The presence of salts diminished the endonuclease activity of pFEN1, with cleavage greatly reduced at 100 mmol/L of NaCl. The activity of pFEN1 was detected only in the presence of magnesium (Mg²⁺) or manganese (Mn²⁺), and pFEN1 showed a higher endonucease activity under the catalysis of Mn²⁺. pFEN1 was thermally stable and had highest activity at temperature range of 60-65℃. Proliferating cell nuclear antigen (PCNA) can significantly promote the activity of pFEN1.[Conclusion] This study confirmed that pFEN1 is a Mg²⁺ or Mn²⁺ dependent endonuclease and PCNA can stimulate its activity.

Key words:thermophilic archaea;Pyrococcus furious;flap endonuclease 1;proliferating cell nuclear antigen (PCNA)