Secreted expression and purification of dengue 2 virus full-length nonstructural glycoprotein NS1 in Pichia pastoris.
The dengue 2 virus (DEN-2) RNA (NGC strain) was used as a substrate to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA followed by polymerase chain reaction amplification of the NS1 region. Products were cloned into pPICZalphaB vector for sequencing and into Pichia pastoris for expression. A recombinant protein with a molecular size of approximately 80 KDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed protein was able to bind with mouse polyclonal antibody or NS1-specific monoclonal antibody of dengue 2 virus. Purified NS1-poly(His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The study also verified that our purified rNS1 protein retained its antigenicity. High-level production of the rNS1 protein up to 70 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length NS1 glycoprotein.
Zhou
Jun-mei
JM
Department of Microbiology, Zhongshan Medical College, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou, 510089, People's Republic of China.
Tang
Yun-xia
YX
Fang
Dan-yun
DY
Zhou
Jing-jiao
JJ
Liang
Yu
Y
Guo
Hui-yu
HY
Jiang
Li-fang
LF
D000330:Aedes / Q000821:virology; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003716:Dengue Virus / Q000235:genetics* / Q000302:isolation & purification*; D015967:Gene Expression Regulation, Viral; D010843:Pichia / Q000821:virology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
C079340:NS1 protein, Dengue virus type 2; D017361:Viral Nonstructural Proteins