DNA-binding property of recombinant capsid protein of Japanese encephalitis virus.
Japanese encephalitis virus (JEV) is a member of the Flaviviridae family, and it is capable of inducing febrile syndrome, encephalitis and death. In this study, the cDNA of JEV-YL capsid (core) protein were cloned and expressed in E. coli. Three expressed recombinant clones (pET32/CORE, pET32/CD and pET32/C2D) including different parts of capsid protein, were constructed from JEV cDNA clone, pJE-S. These recombinant proteins (34, 31, and 26 kDa, respectively) containing an amino terminal tag of six histidines were isolated by the nickel chelate affinity chromatography and the purified products were identified by Western blotting with anti-serum of JEV-infected swine. To examine DNA binding property of the capsid protein, the purified recombinant proteins were assayed by electrophoretic mobility shift assay. The results showed the capsid proteins had the abilities to bind DNA, except the prominent product of pET32/C2D, which had deletion in the middle hydrophobic region of capsid protein and revealed the coding region of the amino acid, F46 to P61, is mediating the DNA binding ability. This study suggests a possible regulatory role for capsid protein in the pathway of JEV infection.
Tseng
Hsi-Nuan
HN
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung 402, Taiwan.
Lee
Chi-Chang
CC
Wong
Min-Liang
ML
Chen
Shu-O
SO
Liu
Jau-Jin
JJ
D001665:Binding Sites; D036022:Capsid Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004664:Encephalitis Virus, Japanese / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D036022:Capsid Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins