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KMID : 0545120170270061098
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Journal of Microbiology and Biotechnology
2017 Volume.27 No. 6 p.1098 ~ p.1105
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VSV-G Viral Envelope Glycoprotein Prepared from Pichia pastoris Enhances Transfection of DNA into Animal Cells
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Liu Xin
Dong Ying
Wang Jingquan
Li Long
Zhong-Zhenmin
Li Yun-Pan
Chen Shao-Jun
Fu Yu-Cai
Xu Wen-Can
Wei Chi-Ju
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Abstract
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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 into Pichia pastoris expression 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 from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.
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KEYWORD
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Vesicular stomatitis virus G glycoprotein, Pichia pastoris, fusion, DNA delivery, large-scale production
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