Expression, purification and functional study of mycobacteriophage D29 histidine-asparagine-histidine endonuclease

Mycobacteriophage histidine-asparagine-histidine endonuclease (mpHNHE) is a protein encoded by mycobacteriophage D29, featuring a conserved HNH motif and belonging to the HNH nuclease superfamily. To explore its physiological functions, the recombinant plasmid pET-28a (+)-mpHNHE was constructed and expressed in E. coli BL21 (DE3). The inclusion body form of the expression product was purified using urea denaturation combined with nickel affinity chromatography and gel filtration chromatography. Structural characterization revealed that mpHNHE exists as a monomer in solution, predominantly composed of β-sheets, and exhibits good structural stability. Enzymatic property studies indicated that mpHNHE has high nuclease activity, significant substrate size selectivity, and metal ion dependence. These findings not only provide new insights into the structure-function relationship of HNH-type nucleases but also provide a molecular basis for the development of new nuclease tools and lay the foundation for understanding the mechanism of mpHNHE in D29.