Generation of a transducible version of a bioactive recombinant human TBX5 transcription factor from E. Coli

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• Protein purification of a recombinant TBX5 fusion protein was performed. • Recombinant TBX5 protein maintains its secondary structure after purification. • Purified TBX5 protein has cell penetration and nuclear translocation potential. • Purified TBX5 protein is bioactive. The protein T-box transcription factor 5 (TBX5) regulates heart and forelimb development in vertebrates, and its deficiency results in Holt-Oram syndrome. Notably, TBX5 functions as a tumor suppressor protein and is a key transcription factor in the reprogramming cocktail used to induce the formation of cardiomyocytes from fibroblasts. Here, we report the expression and purification of a transducible version of recombinant human TBX5 protein from the prokaryotic host Escherichia coli ( E. coli ) under native conditions. First, the protein-coding, codon-optimized TBX5 nucleotide sequence was fused in-frame with three tags (octahistidine, transactivator of transcription, and nuclear localization sequence) at either end of the sequence for affinity purification, cellular uptake, and nuclear delivery. Subsequently, this codon-optimized gene was cloned in a protein expression vector and expressed in E. coli . Various expression parameters were screened to obtain the highest expression of this protein with maximum solubility, to enable purification from the supernatant fraction. Furthermore, the TBX5 fusion protein was purified as a pure (>90%) recombinant protein under native conditions. The purity and identity were confirmed by Coomassie staining and immunoblotting, respectively. Subsequently, the retention of the secondary structure after purification was analyzed and established through far UV circular dichroism spectroscopy. The cellular uptake and nuclear translocation ability of purified TBX5 fusion protein and its biological activity in human cells were demonstrated. The cell-permeant recombinant protein tool established in this study should help elucidate the biological role of human TBX5 in various cellular processes and pave the way for more detailed understanding of cardiac reprogramming and disease-specific studies.