Functional identification of the non-specific nuclease from white spot syndrome virus.
The product encoded by the wsv191 gene from shrimp white spot syndrome virus (WSSV) is homologous with non-specific nucleases (NSN) of other organisms. To functionally identify the protein, the wsv191 gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with 6His-tag at C-terminal. The fusion protein (termed as rWSSV-NSN) was purified using Ni-NTA affinity chromatography under denatured conditions, renatured and characterized by three methods. The results showed that rWSSV-NSN could hydrolyze both DNA and RNA. 5'-RACE result revealed that the transcription initiation site of the wsv191 gene was located at nucleotide residue G of the predicted ATG triplet. Therefore, we concluded that the next ATG should be the genuine translation initiation codon of the wsv191 gene. Western blot analysis revealed that the molecular mass of natural WSSV-NSN was 37 kDa.
Li
Li
L
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, SOA, 178 Daxue Road, Xiamen 361005, P.R. China.
Lin
Shumei
S
Yanga
Feng
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015967:Gene Expression Regulation, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000821:virology*; D012260:Ribonucleases / Q000235:genetics* / Q000302:isolation & purification; D014158:Transcription, Genetic; D046848:White spot syndrome virus 1 / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D012260:Ribonucleases