Soluble expression, purification, and secondary structure determination of human PDX1 transcription factor

Narayan, Gloria; Sundaravadivelu, Pradeep Kumar; Agrawal, Akriti; Gogoi, Ranadeep; Nagotu, Shirisha; Thummer, Rajkumar P.

doi:10.1016/j.pep.2020.105807

Cited by 14 publications

(13 citation statements)

References 47 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%

“…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%

“…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%

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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: Codon Optimization and Cloning Of Human Hand2 Gene Sequencementioning

confidence: 99%

See 1 more Smart Citation

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

Haridhasapavalan

Sundaravadivelu

Mohapatra

et al. 2021

Preprint

Self Cite

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“…An in vitro study of the full-length human PDX1 protein by CD spectroscopy suggested a random coil content of almost 50%. The remaining populations were about equal fractions α-helix, β-sheets, and turns ( 57 ). Although the specific conformational properties of PDX1-N have not yet been described experimentally, amino acid bias in this region and computational disorder prediction tools suggest that this region is not globular, which is consistent with the calculated secondary structure populations determined by CD experiments on the full-length protein ( 57 ).…”

Section: Pdx1 Intrinsically Disordered Regions Orchestrate Protein Fu...mentioning

confidence: 99%

Biophysical insights into glucose-dependent transcriptional regulation by PDX1

Usher

Showalter

2022

Journal of Biological Chemistry

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“…This led to the generation of the HTN‐PDX1 genetic construct and its purification under native conditions from Escherichia coli ( E. coli ). [ 24 ] Here, we demonstrate the biological activity of the purified human recombinant HTN‐PDX1 protein (hereafter, PDX1 fusion protein) using gastric and pancreatic ductal cancer cells.…”

Section: Introductionmentioning

confidence: 99%

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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Soluble expression, purification, and secondary structure determination of human PDX1 transcription factor

Cited by 14 publications

References 47 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

Biophysical insights into glucose-dependent transcriptional regulation by PDX1

Biological activity of recombinant human PDX1 protein produced from Escherichia coli

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