Identification of Optimal Expression Parameters and Purification of a Codon-Optimized Human GLIS1 Transcription Factor from Escherichia coli

Dey, Chandrima; Venkatesan, Vishalini; Thummer, Rajkumar P.

doi:10.1007/s12033-021-00390-z

Cited by 6 publications

(3 citation statements)

References 86 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the present study, we report for the first-time screening and identification of the ideal expression parameters for the soluble expression of rhHAND2 fusion proteins in E. coli . 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 30 – 33 , 39 – 50 . Moreover, we also observed the effect of fusion tags´ position on the expression and production of the quality rhHAND2 fusion protein, similar to our previous observations with ETS2, MESP1, and TBX5 recombinant proteins 30 , 31 , 33 .…”

Section: Discussionsupporting

confidence: 89%

“…It can transduce into the mammalian cells and translocate to its nucleus without a transduction reagent. Similar strategies have been reported earlier by numerous studies for recombinant transcription factors purified from E. coli , namely, OCT4, NANOG, SOX2, ETS2, PDX1, MESP1, GATA4, NGN3, GLIS1, and TBX5 13 , 30 – 33 , 37 , 39 – 43 , 71 , 72 . Our strategy of coupling this transcription factor with the protein transduction domain and NLS facilitated its delivery into the mammalian cell target site.…”

Section: Discussionsupporting

confidence: 60%

“…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%

See 2 more Smart Citations

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

Self Cite

View full text Add to dashboard Cite

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.

show abstract