[Expression and purification of porcine reproductive and respiratory syndrome virus Nsp2 protein and analysis of cleavage activity].
OBJECTIVE
To express and purify Porcine Reproductive and Respiratory Syndrome Virus Nsp2 protein and analyze the protease activity of Nsp2.


METHODS
N-terminus and C-terminus of nsp2 gene were amplified by PCR and inserted into expression vector pET21a(+), respectively. The recombinant protein (Nsp2-N and Nsp2-C) were over expressed in E. coli BL21 and purified by Ni-NTA agarose affinity chromatogram and gel filtration. There is a cysteine protease domain (CP) in Nsp2-N through genetic alignment. In this study, the protease activity of Nsp2-N in cis was analyzed by western blot. Additionally, the predicted peptide substrate were synthesized, the protease activity in trans was analyzed by peptide cleavage assay in vitro.


RESULTS
Two soluble recombinant protein were successfully expressed and purity reached up to 90% after purification. The putative protease domain of Nsp2-N couldn't cleave the predicted substrate through cis cleavage and trans cleavage.


CONCLUSIONS
The putative protease domain in Nsp2-N may need other host factors to act as cofactor, which supply basis for further identification of biological activity and screening of anti-virus drug.
Qu
Hongren
H
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. quhr@im.ac.cn
Li
Yaodong
Y
Hou
Yanhong
Y
Yan
Jinghua
J
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D019316:Porcine respiratory and reproductive syndrome virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase