Characterization of a recombinant thermotolerant argonaute protein as an endonuclease by broad guide utilization

Abstract

Background

Prokaryotic argonaute proteins (pAgos) play an important role in host defense in vivo. Most importantly, the thermophilic pAgos with endonuclease activity hold great potential for programmable genetic manipulation. Therefore, exploring argonaute proteins with unique enzyme properties is desired for understanding their diverse catalytic mechanisms and promoting their applications in biotechnology.

Results

The argonaute protein from archaeon Methanocaldococcus fervens (MfAgo) was cloned and overexpressed in Escherichia coli BL21 (DE3). The recombinant protein showed the expected molecular weight of ~ 85.8 kDa by SDS-PAGE. The activity assays demonstrate that MfAgo has cleavage activities toward single-stranded DNA (ssDNA) targets specifically at the site complementary to the position between nucleotides 10 and 11 of the guide strand. Interestingly, MfAgo utilizes small 5′-phosphorylated ssDNA (5′-P ssDNA), 5′-hydroxylated ssDNA (5′-OH ssDNA), and 5′-phosphorylated ssRNA (5′-P ssRNA) as the guides for catalysis. The optimal temperatures are highly dependent on the type of guide and have a range of 80–90 °C. The addition of 0.5 mM Mn²⁺, Mg²⁺ or Co²⁺ to the reaction system significantly enhanced the enzyme activity. Meanwhile, MfAgo is quite active at NaCl concentrations less than 500 mM. Furthermore, structural modeling analyses suggested that its unique wide guide-dependent activity might be related to differing multiple interactions between guides and the MID domain of MfAgo.

Conclusions

MfAgo shows efficient endonuclease activity for ssDNA cleavage. In contrast to most known pAgos, which recognize only one type of guide, MfAgo uses diverse guides, including 5′-P ssDNA, 5′-OH ssDNA, and 5′-P ssRNA, to specifically cleave targets. Characterization of MfAgo expands the understanding of catalysis in the Ago family and provides clues for future genetic manipulation.

Keywords

Argonaute protein / Nucleic acid guide / Endonuclease / DNA cleavage / Homologous modeling

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Funding

National Natural Science Foundation of China(31770078)

Ministry of Science and Technology(2017YFE0103300)