Cell-penetrating peptide delivery of biologically active oct4 protein into cultured Takifugu rubripes spermary cells

Continuous cell culture of a puffer fish Takifugu rubripes has been established for efficient delivery of exogenous genes or proteins to cultured fish cells. Transcription factor oct4 was chosen for transduction into cultured fish cells because of its conserved structure and function between fish and mammals. In this work, the T. rubripes oct4 gene was cloned and expressed in Escherichia coli as a recombinant protein by introducing cell-penetrating peptide (CPP) poly-arginine (11R) and 6His-tag at the C-terminus. After purification, recombinant proteins were added to the growth medium and incubated with T. rubripes spermary cells. Recombinant proteins that crossed the cell membrane were detected in the cytoplasm and nucleus by western blot and immunofluorescent observation. The function of transduced oct4 as a transcription factor in fish cells was confirmed by driving green fluorescent protein expression in the pEGFP-1 reporter construct with the conserved specific oct4-binding sequence from mouse Mus musculus. Taken together, 11R can be an efficient CPP in delivering fusion proteins to cultured fish cells. (C) 2014 The Fisheries Society of the British Isles