title: Secreted expression of dengue virus type 2 full-length envelope glycoprotein in Pichia pastoris.
abstract: The full-length envelope glycoprotein gene of dengue virus type 2 was cloned using an RT-PCR method from the infected C6/36 cells and inserted into pPICZaB vector. The recombinant plasmid was integrated into Pichia pastoris by electroporation and the expressed product was identified by SDS-PAGE and Western blotting. High-level secreted expression was performed by determining the Mut(+) phenotype and screening multi-copy integrants in the recombinant yeast cells. A recombinant protein with a molecular size of approximately 69 kDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed supernatant was able to bind with mouse polyclonal antibody or E-specific monoclonal antibody of dengue-2 virus. Purified E-poly (His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The results of Western blotting and solid-phase ELISA using dengue virus antibodies indicated that the purified recombinant E glycoprotein retained its antigenicity. High-level production of the recombinant E protein up to 100 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length envelope glycoprotein.
authors.affiliation: Department of Microbiology, Zhongshan Medical College, Sun Yat-Sen University, 74 Zhongshan Road II, Guangzhou 510089, People's Republic of China. Hywei2002@yahoo.com
authors.forename: Hui-yong
authors.initials: HY
authors.lastname: Wei
authors.forename: Li-fang
authors.initials: LF
authors.lastname: Jiang
authors.forename: Yao-hua
authors.initials: YH
authors.lastname: Xue
authors.forename: Dan-yun
authors.initials: DY
authors.lastname: Fang
authors.forename: Hui-yu
authors.initials: HY
authors.lastname: Guo
mesh_terms: D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism*; D018274:Electroporation; D015870:Gene Expression / Q000187:drug effects; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D000432:Methanol / Q000494:pharmacology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D000432:Methanol