title: VSV-G Viral Envelope Glycoprotein Prepared fromPichia pastorisEnhances Transfection of DNA into Animal Cells.
abstract: Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag intoPichia pastorisexpression vector pPIC3.5K. Three clones (Muts) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced fromP. pastoriswith biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China.
authors.forename: Jingquan
authors.initials: J
authors.lastname: Wang
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China.
authors.forename: Long
authors.initials: L
authors.lastname: Li
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China.
authors.forename: Zhenmin
authors.initials: Z
authors.lastname: Zhong
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China.
authors.forename: Yun-Pan
authors.initials: YP
authors.lastname: Li
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China.
authors.forename: Shao-Jun
authors.initials: SJ
authors.lastname: Chen
authors.affiliation: Laboratory of Cell Senescence, Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.
authors.forename: Yu-Cai
authors.initials: YC
authors.lastname: Fu
authors.affiliation: Department of Endocrinology, the First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.
authors.forename: Wen-Can
authors.initials: WC
authors.lastname: Xu
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China.
authors.forename: Chi-Ju
authors.initials: CJ
authors.lastname: Wei
mesh_terms: D000818:Animals; D002452:Cell Count; D004247:DNA; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D010843:Pichia / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D011523:Protoplasts; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D004247:DNA
keywords: DNA delivery; Pichia pastoris; Vesicular stomatitis virus G glycoprotein; fusion; large-scale production