Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli.
Krishna Kumar HaridhasapavalanPradeep Kumar SundaravadiveluSrirupa BhattacharyyaSujal Harsh RanjanKhyati RainaRajkumar P ThummerPublished in: Bioprocess and biosystems engineering (2021)
Transcription factor GATA4 is expressed during early embryogenesis and is vital for proper development. In addition, it is a crucial reprogramming factor for deriving functional cardiomyocytes and was recently identified as a tumor suppressor protein in various cancers. To generate a safe and effective molecular tool that can potentially be used in a cell reprogramming process and as an anti-cancer agent, we have identified optimal expression parameters to obtain soluble expression of human GATA4 in E. coli and purified the same to homogeneity under native conditions using immobilized metal ion affinity chromatography. The identity of GATA4 protein was confirmed using western blotting and mass spectrometry. Using circular dichroism spectroscopy, it was demonstrated that the purified recombinant protein has maintained its secondary structure, primarily comprising of random coils and α-helices. Subsequently, this purified recombinant protein was applied to human cells and was found that it was non-toxic and able to enter the cells as well as translocate to the nucleus. Prospectively, this cell- and nuclear-permeant molecular tool is suitable for cell reprogramming experiments and can be a safe and effective therapeutic agent for cancer therapy.
Keyphrases
- transcription factor
- mass spectrometry
- single cell
- cell therapy
- binding protein
- escherichia coli
- poor prognosis
- cancer therapy
- stem cells
- high resolution
- protein protein
- endothelial cells
- induced apoptosis
- liquid chromatography
- drug delivery
- oxidative stress
- single molecule
- mesenchymal stem cells
- high speed
- functional connectivity
- cell free
- signaling pathway
- young adults