Expression of recombinant insulin-like growth factor-binding protein-3 receptor in mammalian cell line and prokaryotic (Escherichia coli) expression systems

Document Type : Original Article


1 Department of Clinical Biochemistry, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran


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.


Joyce S, Nour AM. Blocking transmembrane219 protein signaling inhibits autophagy and restores normal cell death. PLoS One 2019;14:1-25.  Back to cited text no. 1
Ingermann AR, Yang YF, Han J, Mikami A, Garza AE, Mohanraj L, et al. Identification of a novel cell death receptor mediating IGFBP-3-induced anti-tumor effects in breast and prostate cancer. Biol Chem 2010;285:30233-46.  Back to cited text no. 2
Cai Q, Dozmorov M, Oh Y. IGFBP-3/IGFBP-3 receptor system as an anti-tumor and anti-metastatic signaling in cancer. Cells 2020;9:1261.  Back to cited text no. 3
Baxter RC. Insulin-like growth factor binding protein-3 (IGFBP-3): Novel ligands mediate unexpected functions. J Cell Commun Signal 2013;7:179-89.  Back to cited text no. 4
Han J, Jogie-Brahim S, Harada A, Oh Y. Insulin-like growth factor-binding protein-3 suppresses tumor growth via activation of caspase-dependent apoptosis and cross-talk with NF-κB signaling. Cancer Lett 2011;307:200-10.  Back to cited text no. 5
Mohanraj L, Oh Y. Targeting IGF-I, IGFBPs and IGF-I receptor system in cancer: The current and future in breast cancer therapy. Recent Pat Anticancer Drug Discov 2011;6:166-77.  Back to cited text no. 6
Shahjee HM, Bhattacharyya N. Activation of various downstream signaling molecules by IGFBP-3. J Cancer Ther 2014;5:830-5.  Back to cited text no. 7
Lee YC, Jogie-Brahim S, Lee DY, Han J, Harada A, Murphy LJ, et al. Insulin-like growth factor-binding protein-3 (IGFBP-3) blocks the effects of asthma by negatively regulating NF-κB signaling through IGFBP-3R-mediated activation of caspases. J Biol Chem 2011;286:17898-909.  Back to cited text no. 8
Johnsen SP, Hundborg HH, Sørensen HT, Orskov H, Tjønneland A, Overvad K, et al. Insulin-like growth factor (IGF) I, -II, and IGF binding protein-3 and risk of ischemic stroke. J Clin Endocrinol Metab 2005;90:5937-41.  Back to cited text no. 9
Chang KH, Chan-Ling T, McFarland EL, Afzal A, Pan H, Baxter LC, et al. IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development. Proc Natl Acad Sci U S A 2007;104:10595-600.  Back to cited text no. 10
Ikonen M, Liu B, Hashimoto Y, Ma L, Lee KW, Niikura T, et al. Interaction between the Alzheimer's survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis. Proc Natl Acad Sci U S A 2003;100:13042-7.  Back to cited text no. 11
Mehta HH, Gao Q, Galet C, Paharkova V, Wan J, Said J, et al. IGFBP-3 is a metastasis suppression gene in prostate cancer. Cancer Res 2011;71:5154-63.  Back to cited text no. 12
Ansari A, Gheysarzadeh A, Mofid MR. The Interaction of Insulin-Like Growth Factor Binding Protein 3 (IGFBP-3) in Insulin-Like Growth Factor (IGF)-Independent System. Journal of Isfahan Medical School, 2017;35:1452–61.  Back to cited text no. 13
Harada A, Jogie-Brahim S, Oh Y. Tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone suppresses a newly identified anti-tumor IGFBP-3/IGFBP-3R system in lung cancer cells. Lung Cancer 2013;80:270-7.  Back to cited text no. 14
Gheysarzadeh A, Bakhtiari H, Ansari A, Emami Razavi A, Emami MH, Mofid MR. The insulin-like growth factor binding protein-3 and its death receptor in pancreatic ductal adenocarcinoma poor prognosis. J Cell Physiol 2019;234:23537-46.  Back to cited text no. 15
Mofid MR, Gheysarzadeh A, Bakhtiyari S. Insulin-like growth factor binding protein 3 chemosensitizes pancreatic ductal adenocarcinoma through its death receptor. Pancreatology 2020;20:1442-50.  Back to cited text no. 16
D'Addio F, La Rosa S, Maestroni A, Jung P, Orsenigo E, Ben Nasr M, et al. Circulating IGF-I and IGFBP3 levels control human colonic stem cell function and are disrupted in diabetic enteropathy. Cell Stem Cell 2015;17:486-98.  Back to cited text no. 17
Cheng CW, Yilmaz ÖH. In translation IGFBP3 and T1D: Systemic factors in colonic stem cell function and diabetic enteropathy. Cell Stem Cell 2015;17:379-80.  Back to cited text no. 18
Yin J, Li G, Ren X, Herrler G. Select what you need: A comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol 2007;127:335-47.  Back to cited text no. 19
Kubick S, Gerrits M, Merk H, Stiege W, Erdmann VA. In vitro synthesis of posttranslationally modified membrane proteins. Curr Top Membr 2009;63:25-49.  Back to cited text no. 20
Bhatt FH, Jeffery CJ. Expression, detergent solubilization, and purification of a membrane transporter, the MexB multidrug resistance protein. J Vis Exp 2010;3: 2134.  Back to cited text no. 21
Fowlkes JL, Thrailkill KM, Serra DM, Suzuki K, Nagase H. Matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases. Prog Growth Factor Res 1995;6:255-63.  Back to cited text no. 22
Yamanaka Y, Fowlkes JL, Wilson EM, Rosenfeld RG, Oh Y. Characterization of insulin-like growth factor binding protein-3 (IGFBP-3) binding to human breast cancer cells: Kinetics of IGFBP-3 binding and identification of receptor binding domain on the IGFBP-3 molecule. Endocrinology 1999;140:1319-28.  Back to cited text no. 23
Lee CM, He CH, Nour AM, Zhou Y, Ma B, Park JW, et al. IL-13Rα2 uses TMEM219 in chitinase 3-like-1-induced signalling and effector responses. Nat Commun 2016;7:1-2.  Back to cited text no. 24
He CH, Lee CG, Dela CC, Lee CM, Zhou Y, Ahangari F, et al. Chitinase 3-like 1 regulates cellular and tissue responses via IL-13 receptor α2. Cell Rep 2013;4:830-41.  Back to cited text no. 25
Burkhart BJ, Schwalen CJ, Mann G, Naismith JH, Mitchell DA. YcaO-dependent posttranslational amide activation: Biosynthesis, structure, and function. Chem Rev 2017;117:5389-456.  Back to cited text no. 26
Walsh G, Jefferis R. Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 2006;24:1241-52.  Back to cited text no. 27
Smith SM. Strategies for the purification of membrane proteins. Methods Mol Biol. 2017;1485: 389-400.  Back to cited text no. 28
Zhang X, Miller KW. Dodecyl maltopyranoside enabled purification of active human GABA type A receptors for deep and direct proteomic sequencing. Mol Cell Proteomics 2015;14:724-38.  Back to cited text no. 29