Soluble expression, purification, and secondary structure determination of human MESP1 transcription factor

Haridhasapavalan, Krishna Kumar; Ranjan, Sujal Harsh; Bhattacharyya, Srirupa; Thummer, Rajkumar P.

doi:10.1007/s00253-021-11194-1

Cited by 16 publications

(10 citation statements)

References 64 publications

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“…As a transcription factor, it has its major role inside the nucleus; therefore, its nuclear entry is one of the most critical aspects of its functionality. Similar fusion strategies were employed in previous studies, including us for the efficient subcellular and subnuclear delivery of reprogramming factors such as OCT4, NANOG, SOX2, PDX1, and GATA4 in the form of recombinant proteins in mammalian cells [29,33,35,[64][65][66][67]. These studies have used TAT and NLS as fusion tags to efficiently deliver protein of interest into the cell and its nucleus, respectively, to exert their biological function in mammalian cells [13,35,64,65].…”

Section: Discussionmentioning

confidence: 93%

“…The protein concentration of sonicated and purified samples was estimated using Bradford assay [32]. The SDS-PAGE and immunoblotting were performed as described in our recent studies [29,30]. The antibodies used and their respective concentrations are listed in Table S5.…”

Section: Sds-page and Immunoblottingmentioning

confidence: 99%

“…Far-ultraviolet (UV) circular dichroism (CD) spectroscopy was carried out as described in our recent studies [29,30]. From the CD spectrum obtained, the secondary structure content of the purified HAND2 protein was estimated using the online tool, Beta Structure Selection (BeStSel; http://bestsel.elte.hu/index.php).…”

Section: Far-ultraviolet Circular Dichroism Spectroscopymentioning

confidence: 99%

“…Immunocytochemistry and microscopy were performed as described recently [29,33]. For immunocytochemistry, the antibodies and their respective concentrations used are listed in Table S5.…”

Section: Immunocytochemistry and Microscopymentioning

confidence: 99%

“…Recently, we have demonstrated the heterologous expression and purification of human cardiac reprogramming factors, namely GATA4 [29], ETS2 [30], and MESP1 [31], in recombinant forms. Here, we demonstrated the soluble expression and purification of recombinant human HAND2 protein from E. coli under native conditions having efficient cell permeability and nuclear translocation ability.…”

Section: Introductionmentioning

confidence: 99%

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Generation of transducible version of a recombinant human HAND2 transcription factor from Escherichia coli

Haridhasapavalan

Sundaravadivelu

Mohapatra

et al. 2021

Preprint

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Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. Also, 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 (E. coli) strain, BL21(DE3). Next, the effect (in terms of expression) of tagging of fusion tags with this recombinant protein at two different terminals was also investigated. Notably, using affinity chromatography, we established the one-step homogeneous purification of human recombinant HAND2 protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. Furthermore, we show that this purified human protein could transduce the human cells and translocate to its nucleus. Prospectively, the purified recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.

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Section: Discussionmentioning

confidence: 93%

Section: Sds-page and Immunoblottingmentioning

confidence: 99%

Section: Far-ultraviolet Circular Dichroism Spectroscopymentioning

confidence: 99%

“…Immunocytochemistry and microscopy were performed as described recently [29,33]. For immunocytochemistry, the antibodies and their respective concentrations used are listed in Table S5.…”

Section: Immunocytochemistry and Microscopymentioning

confidence: 99%