Generation of a recombinant version of a biologically active cell-permeant human HAND2 transcription factor from E. coli

Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. It is one of the key cardiac factors crucial for the enhanced derivation of functional and mature myocytes from non-myocyte cells. Here, we report the generation of the recombinant human HAND2 fusion protein from the heterologous system. First, we cloned the full-length human HAND2 gene (only protein-coding sequence) after codon optimization along with the fusion tags (for cell penetration, nuclear translocation, and affinity purification) into the expression vector. We then transformed and expressed it in Escherichia coli strain, BL21(DE3). Next, the effect (in terms of expression) of tagging fusion tags with this recombinant protein at two different terminals was also investigated. Using affinity chromatography, we established the one-step homogeneous purification of recombinant human HAND2 fusion protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. We then showed that this purified human protein could transduce the human cells and translocate to its nucleus. The generated recombinant HAND2 fusion protein showed angiogenic potential in the ex vivo chicken embryo model. Following transduction in MEF2C overexpressing cardiomyoblast cells, this purified recombinant protein synergistically activated the alpha-MHC promoter and induced GFP expression in the alpha-MHC-eGFP reporter assay. Prospectively, the purified bioactive recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.