Expression of Recombinant Insulin‑Like Growth Factor‑Binding Protein‑3 Receptor in Mammalian Cell Line and Prokaryotic (Escherichia coli) Expression Systems

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Article Type:
Research/Original Article (دارای رتبه معتبر)
Abstract:
Background

Insulin‑like growth factor binding protein‑3 receptor (IGFBP‑3R) (Transmembrane protein 219 [TMEM219]) binds explicitly to IGFBP‑3 and exerts its apoptotic and autophagy signalling pathway. Constructing a Henrietta Lacks (HeLa) h6‑TMEM219 cell characterize the therapeutic potent of TMEM219 that could interrupt the IGFBP‑3/TMEM219 pathway, in cancer treatment and destructive cell illnesses such as diabetes and Alzheimer’s.

Materials and Methods

First, to develop stable overexpressed HeLa h6‑TMEM219 cells, and Escherichia coli BL21 (DE3) with high IGFBP‑3R expression, the purchased pcDNA3.1‑h6‑TMEM219 plasmid was transformed and integrated using CaCl2 and chemical transfection reagents, respectively. The pcDNA3.1‑h6‑TMEM219 transfection and protein expression was evaluated by the polymerase chain reaction (PCR), western blotting, and flow cytometry. Following the induction of h6‑TMEM219 expression, a protein was purified using Ni‑NTA chromatography and evaluated by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‑PAGE).

Results

The 606 base pairs sequence in PCR outcomes confirmed successful pcDNA3.1‑h6‑TMEM219 transformation in E. coli BL21 and integration into the HeLa genome. The analysis of protein samples from induced E. coli BL21 and purified protein demonstrate a band of approximately 22 kDa on SDS‑PAGE. Moreover, besides western blot analysis, flow cytometry findings illustrate approximately 84% of transfected HeLa cells (HeLa h6‑TMEM219) overexpressed h6‑TMEM219 on their surface.

Conclusion

We designed a new experiment in the h6‑TMEM219 expression procedure in both eukaryotic and prokaryotic hosts. All of our results confirm appropriate transformation and transfection and importantly, approve h6‑TMEM 219 membrane expression. Finally, the HeLa h6‑TMEM219 cells and the newly purified h6‑TMEM219 leverage new studies for molecular diagnostic studies and characterize the therapeutic agents against IGFBP‑3/TMEM219 signalling pathway in devastating illnesses in vitro and in vivo.

Language:
English
Published:
Advanced Biomedical Research, Volume:12 Issue: 2, Feb 2022
Page:
19
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