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Efficient Production of Cell-permeable Oct4 Protein Using 30Kc19 Protein Originating from Silkworm

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  • Protein Engineering and Enzyme Technology
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Abstract

Oct4 is a transcription factor (TF), which works as a transcriptional activator and plays an essential role in reprogramming or direct conversion of somatic cells. Many strategies have been reported to transduce TFs including Oct4 into the cells by delivering the gene; however, the reported methods are associated with several problems such as genetic mutation, cytotoxicity, and low transfection efficiency. Although direct delivery of the transcription factors has been suggested as an alternative method, its use is limited due to poor solubility and stability of recombinant TFs. Previously, it has been reported that the conjugation of Bombyx mori 30Kc19 protein not only facilitates intracellular delivery but also augments soluble expression in Escherichia coli and stability of recombinant TFs. However, the expression levels of the 30Kc19-conjugated TFs were still low. In the present work, we reconstituted 30Kc19-conjugated Oct4 protein (Oct4-30Kc19) expressed as inclusion bodies and compared to soluble expression. The insoluble Oct4-30Kc19 was denatured and refolded in an affinity chromatography column. The refolded Oct4-30Kc19 exhibited cell-penetrating property without any significant cytotoxicity and enhancement in thermal stability similar to soluble Oct4-30Kc19; while a drastic increase in total production yield was observed. In addition, the transcriptional activity of the refolded Oct4-30Kc19 was demonstrated through Oct4-GFP reporter assay. It is anticipated that the refolded Oct4-30Kc19 can be utilized as a valuable tool for transgene-free cellular reprogramming and direct conversion.

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Acknowledgments

This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF- 2017M3A9C6031798) and the Ministry of Education (NRF- 2018R1D1A1B07050422). This study was also supported by 2017 Research Grant from Kangwon National University (No. 520170405).

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Authors and Affiliations

  1. Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, 24341, Korea

    Jaeyoung Lee & Ju Hyun Park

  2. Department of Biotechnology and Bioengineering, Kangwon National University, Chuncheon, 24341, Korea

    Hee Ho Park

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  1. Jaeyoung Lee
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Lee, J., Park, H.H. & Park, J.H. Efficient Production of Cell-permeable Oct4 Protein Using 30Kc19 Protein Originating from Silkworm. Biotechnol Bioproc E 24, 964–971 (2019). https://doi.org/10.1007/s12257-019-0204-5

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