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Synthesis in Escherichia coli cells and characterization of the active exoribonuclease of severe acute respiratory syndrome coronavirus



Synthesis in Escherichia coli cells and characterization of the active exoribonuclease of severe acute respiratory syndrome coronavirus



Molekuliarnaia Biologiia 43(3): 446-454



The nsp14 protein, an exoribonuclease of the DEDD superfamily encoded by severe acute respiratory syndrome coronavirus (SARS-CoV), was expressed in fusion with different affinity tags. The recombinant nspl4 proteins with either GST fusion or 6-histidine tag were shown to possess ribonuclease activity but nspl4 with a short MGHHHHHHGS tag sequence at the N-terminus increased the solubility of nspl4 protein and facilitated the protein purification. Mutations of the conserved residues of nspl4 resulted in significant attenuation but not abolishment of the ribonuclease activity. Combination of fluorescence and circular dichroism spectroscopy analyses showed that the conformational stability of nsp14 protein varied with many external factors such as pH, temperature and presence of denaturing chemicals. These results provide new information on the structural features and would be helpful for further characterization of this functionally important protein.

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Accession: 056073861

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PMID: 19548531


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