Protein Production and Purification of a Codon-Optimized Human NGN3 Transcription Factor from E. coli

Narayan, Gloria; Agrawal, Akriti; Joshi, Neha; Gogoi, Ranadeep; Nagotu, Shirisha; Thummer, Rajkumar P.

doi:10.1007/s10930-021-10020-x

Cited by 8 publications

(6 citation statements)

References 75 publications

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“…As a transcription factor, it has a 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 ours, for the efficient subcellular and subnuclear delivery of reprogramming factors such as OCT4, NANOG, SOX2, GATA4, NGN3, and TBX5 in the form of recombinant proteins in mammalian cells 32 , 33 , 37 , 42 , 43 , 71 – 74 . 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 , 32 , 33 , 37 , 42 , 43 , 71 , 72 .…”

Section: Discussionmentioning

confidence: 86%

“…It has a high transformation efficiency and is economical for recombinant protein production in large quantities, fast growth, and well-known genetics 61 , 62 . In addition, this organism is commonly used to produce human recombinant proteins that do not require posttranslational modifications for functionality 33 , 39 , 42 , 43 , 63 – 68 . Phosphorylation of HAND2 by Akt leads to the formation of the heterodimer, thereby inhibiting its transcriptional activity 69 .…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 86%

Section: Discussionmentioning

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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“…Thus, the addition of TAT and NLS at the N‐terminus end of the PDX1 protein has proved beneficial for effective cell penetration and nuclear translocation in mammalian cells. Several other studies also reported TAT‐ and NLS‐mediated translocation of other transcription factors, [ 24–26,29–31 ] deriving desired cells for biological applications. [ 26,30,31 ]…”

Section: Resultsmentioning

confidence: 99%

“…RNA isolation, along with complementary DNA (cDNA) preparation and quantitative reverse transcription polymerase chain reaction (RT‐qPCR), was performed as mentioned earlier. [ 25 ] Briefly, PANC‐1 cells were plated (1 × 10 5 cells each well) and treated with 200 and 400 nM of PDX1 fusion protein for five consecutive days. Posttreatment, total RNA of the treated as well as untreated cells were isolated using TRIzol reagent (Invitrogen), and subsequently, cDNA synthesis was performed.…”

Section: Methodsmentioning

confidence: 99%

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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 Recombinant Version of a Bioactive Human MEF2C Transcription Factor from E. coli

Haridhasapavalan,

Sundaravadivelu,

Voorkara

et al. 2023

Healthcare Research and Related Technologies

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Protein Production and Purification of a Codon-Optimized Human NGN3 Transcription Factor from E. coli

Cited by 8 publications

References 75 publications

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

Generation of Recombinant Version of a Bioactive Human MEF2C Transcription Factor from E. coli

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