Generation of biologically active recombinant human OCT4 protein from E. coli

Octamer-binding transcription factor 4 (OCT4) is vital for early embryonic development and is a master regulator of pluripotency in embryonic stem cells. Notably, OCT4 is a key reprogramming factor to derive induced pluripotent stem cells, which have tremendous prospects in regenerative medicine. In the current study, we report heterologous expression and purification of human OCT4 in E. coli to produce pure recombinant protein under native conditions. To achieve this, the 1083 bp coding sequence of the human OCT4 gene was codon-optimized for heterologous expression in E. coli. The codon-optimized sequence was fused with fusion tags, namely a cell-penetrating peptide sequence for intracellular delivery, a nuclear localization sequence for intranuclear delivery, and a His-tag for affinity purification. Subsequently, the codon-optimized sequence and the fusion tags were cloned in the protein expression vector, pET28a(+), and transformed into E. coli strain BL21(DE3) for expression. The recombinant OCT4 protein was purified from the soluble fraction under native conditions using immobilized metal ion affinity chromatography in a facile manner, and its identity was confirmed by Western blotting and mass spectrometry. Furthermore, the secondary structure of the recombinant protein was analyzed using far ultraviolet circular dichroism spectroscopy, which confirmed that the purified fusion protein maintained a secondary structure conformation, and it predominantly composed of α-helices. Next, the recombinant OCT4 protein was applied to human cells, and was found that it was able to enter the cells and translocate to the nucleus. Furthermore, the biological activity of the transduced OCT4 protein was also demonstrated on human cells. This recombinant tool can substitute for genetic and viral forms of OCT4 to enable the derivation of integration-free pluripotent cells. It can also be used to elucidate its biological role in various cellular processes and diseases and for structural and biochemical studies.