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

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.