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 proper...
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doaj-98d45f291ec7476d9a95a1a92a9293872020-11-25T03:26:19ZengSpringerOpenBioresources and Bioprocessing2197-43652019-06-016111010.1186/s40643-019-0254-8Characterization of a recombinant thermotolerant argonaute protein as an endonuclease by broad guide utilizationYuesheng Chong0Qian Liu1Fei Huang2Dong Song3Yan Feng4State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong UniversityState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong UniversityState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong UniversityState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong UniversityState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong UniversityAbstract 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.http://link.springer.com/article/10.1186/s40643-019-0254-8Argonaute proteinNucleic acid guideEndonucleaseDNA cleavageHomologous modeling |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yuesheng Chong Qian Liu Fei Huang Dong Song Yan Feng |
spellingShingle |
Yuesheng Chong Qian Liu Fei Huang Dong Song Yan Feng Characterization of a recombinant thermotolerant argonaute protein as an endonuclease by broad guide utilization Bioresources and Bioprocessing Argonaute protein Nucleic acid guide Endonuclease DNA cleavage Homologous modeling |
author_facet |
Yuesheng Chong Qian Liu Fei Huang Dong Song Yan Feng |
author_sort |
Yuesheng Chong |
title |
Characterization of a recombinant thermotolerant argonaute protein as an endonuclease by broad guide utilization |
title_short |
Characterization of a recombinant thermotolerant argonaute protein as an endonuclease by broad guide utilization |
title_full |
Characterization of a recombinant thermotolerant argonaute protein as an endonuclease by broad guide utilization |
title_fullStr |
Characterization of a recombinant thermotolerant argonaute protein as an endonuclease by broad guide utilization |
title_full_unstemmed |
Characterization of a recombinant thermotolerant argonaute protein as an endonuclease by broad guide utilization |
title_sort |
characterization of a recombinant thermotolerant argonaute protein as an endonuclease by broad guide utilization |
publisher |
SpringerOpen |
series |
Bioresources and Bioprocessing |
issn |
2197-4365 |
publishDate |
2019-06-01 |
description |
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. |
topic |
Argonaute protein Nucleic acid guide Endonuclease DNA cleavage Homologous modeling |
url |
http://link.springer.com/article/10.1186/s40643-019-0254-8 |
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