title: Secreted expression and purification of dengue 2 virus full-length nonstructural glycoprotein NS1 in Pichia pastoris.
abstract: 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.
authors.affiliation: Department of Microbiology, Zhongshan Medical College, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou, 510089, People's Republic of China.
authors.forename: Jun-mei
authors.initials: JM
authors.lastname: Zhou
authors.forename: Yun-xia
authors.initials: YX
authors.lastname: Tang
authors.forename: Dan-yun
authors.initials: DY
authors.lastname: Fang
authors.forename: Jing-jiao
authors.initials: JJ
authors.lastname: Zhou
authors.forename: Yu
authors.initials: Y
authors.lastname: Liang
authors.forename: Hui-yu
authors.initials: HY
authors.lastname: Guo
authors.forename: Li-fang
authors.initials: LF
authors.lastname: Jiang
mesh_terms: 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*
chemical_list: C079340:NS1 protein, Dengue virus type 2; D017361:Viral Nonstructural Proteins