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 in...

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Bibliographic Details
Published in:Virus genes 2006-08, Vol.33 (1), p.27-32
Main Authors: Zhou, Jun-mei, Tang, Yun-xia, Fang, Dan-yun, Zhou, Jing-jiao, Liang, Yu, Guo, Hui-yu, Jiang, Li-fang
Format: Article
Language:English
Subjects:
Aedes - virology
Animals
Antigenicity
Cell Line
Cloning, Molecular
Complementary DNA
Dengue fever
Dengue virus
Dengue Virus - genetics
Dengue Virus - isolation & purification
Dengue virus type 2
Fusion protein
Gene Expression Regulation, Viral
Glycoproteins
Monoclonal antibodies
Pichia - virology
Pichia pastoris
Proteins
RNA viruses
RNA-directed DNA polymerase
Viral Nonstructural Proteins - genetics
Viral Nonstructural Proteins - isolation & purification
Viral Nonstructural Proteins - metabolism
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Summary: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.
ISSN:0920-8569
1572-994X
DOI:10.1007/s11262-005-0036-6