Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli

Haridhasapavalan, Krishna Kumar; Sundaravadivelu, Pradeep Kumar; Bhattacharyya, Srirupa; Ranjan, Sujal Harsh; Raina, Khyati; Thummer, Rajkumar P.

doi:10.1007/s00449-021-02516-8

Cited by 16 publications

(13 citation statements)

References 49 publications

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“…Consistent with this, we and others have previously demonstrated the effect of expression parameters on the expression, solubility, stability, and secondary structure conformation of recombinant proteins [29-31, 33, 34, 37-43]. Moreover, we also observed the effect of tagging fusion tags at either terminal in the expression and production of the quality recombinant HAND2 fusion protein, similar to our previous observations with ETS2 and MESP1 recombinant proteins [30,31]. To the best of our knowledge, this is the first study to establish a one-step purification to obtain a highly pure recombinant human HAND2 fusion protein under native (from soluble cell fraction) conditions that has its secondary structure retained.…”

Section: Discussionsupporting

confidence: 92%

“…The cells were transformed with appropriate recombinant plasmids harboring the HAND2 fusion gene and cultured as described recently [31]. Subsequently, the culture (20 mL) was induced with a required concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (HiMedia) and incubated for a respective time at a respective temperature depending on experimental requirements.…”

Section: Identification Of Ideal Expression Parametersmentioning

confidence: 99%

“…2a and 2b (top)]. Various studies have reported that the expression, solubility, and stability of human proteins heterologously expressed in E. coli is influenced by the position of the fusion tags [29][30][31][33][34][35][36]. These fusion gene inserts (HTN-HAND2 and HAND2-NTH) were artificially synthesized and subcloned into the pET28a(+) expression vector containing a tightly regulated T7 promoter (inducible).…”

Section: Codon Optimization and Cloning Of Human Hand2 Gene Sequencementioning

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: Discussionsupporting

confidence: 92%

Section: Identification Of Ideal Expression Parametersmentioning

confidence: 99%

Section: Codon Optimization and Cloning Of Human Hand2 Gene Sequencementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Haridhasapavalan

Sundaravadivelu

Mohapatra

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recently, we have demonstrated the heterologous expression and purification of human cardiac reprogramming factors, namely ETS2 30 , MESP1 31 , GATA4 32 , and TBX5 33 , in recombinant forms. Here, we have demonstrated the soluble expression and purification of recombinant human HAND2 (rhHAND2) protein from E. coli under native conditions having efficient cell permeability, nuclear translocation ability, and angiogenic potential.…”

Section: Introductionmentioning

confidence: 99%

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

Haridhasapavalan

Sundaravadivelu

Joshi

et al. 2022

Sci Rep

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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 α-MHC promoter and induced GFP expression in the α-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.

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Biological activity of recombinant human PDX1 protein produced from Escherichia coli

Narayan,

Sen,

Nagotu

et al. 2023

J Biochem & Molecular Tox

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Pancreatic and duodenum homeobox 1 (PDX1) is considered as a pivotal transcription factor that acts as a “master regulator” in pancreatogenesis and maintenance of β‐cells. Earlier study has reported that PDX1 also functions as a tumor suppressor in human gastric cancer cells by inhibiting cell growth. Here, we report the bioactivity of the purified human PDX1 fusion protein using various assays like cell migration, proliferation, cell cycle analysis, and gene expression. In cancer cells, recombinant PDX1 protein reduced cell migration and proliferation, and arrested cell growth by inducing apoptosis in gastric cancer cells. In pancreatic ductal cancer cells, the application of the PDX1 protein resulted in the induction of insulin gene expression. The results of these experiments demonstrate the biological activity imparted by recombinant human PDX1 fusion protein on gastric and pancreatic cancer cells and its usefulness as a biological tool to elucidate its function in various cellular processes.

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Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli

Cited by 16 publications

References 49 publications

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

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

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

Biological activity of recombinant human PDX1 protein produced from Escherichia coli

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