Secreted expression of dengue virus type 2 full-length envelope glycoprotein in Pichia pastoris.
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.
Wei
Hui-yong
HY
Department of Microbiology, Zhongshan Medical College, Sun Yat-Sen University, 74 Zhongshan Road II, Guangzhou 510089, People's Republic of China. Hywei2002@yahoo.com
Jiang
Li-fang
LF
Xue
Yao-hua
YH
Fang
Dan-yun
DY
Guo
Hui-yu
HY
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
D006023:Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D000432:Methanol