Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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VSV-G Viral Envelope Glycoprotein Prepared from Pichia pastoris Enhances Transfection of DNA into Animal Cells

  • Liu, Xin (Multidisciplinary Research Center, Shantou University) ;
  • Dong, Ying (Multidisciplinary Research Center, Shantou University) ;
  • Wang, Jingquan (Multidisciplinary Research Center, Shantou University) ;
  • Li, Long (Multidisciplinary Research Center, Shantou University) ;
  • Zhong, Zhenmin (Multidisciplinary Research Center, Shantou University) ;
  • Li, Yun-Pan (Multidisciplinary Research Center, Shantou University) ;
  • Chen, Shao-Jun (Multidisciplinary Research Center, Shantou University) ;
  • Fu, Yu-Cai (Laboratory of Cell Senescence, Shantou University Medical College) ;
  • Xu, Wen-Can (Department of Endocrinology, the First Affiliated Hospital of Shantou University Medical College) ;
  • Wei, Chi-Ju (Multidisciplinary Research Center, Shantou University)
  • Received : 2016.12.20
  • Accepted : 2017.03.15
  • Published : 2017.06.28
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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 into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) 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.

Keywords

References

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