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Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli.

ABSTRACT: BACKGROUND:The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this research we cloned and expressed the full-length IGFBP3 in the pET-11a vector and BL21 (DE3) expression host. MATERIALS AND METHODS:First the sequence encoding of IGFBP3 was designed based on the amino acid sequence of the protein and then by codon optimization, in order to ensure the maximum expression in Escherichia coli. In the next step, the synthetic DNA encoding IGFBP3 was inserted into the pUC57 vector, at the appropriate restriction sites and then subcloned in the pET-11a expression vector in the same restriction sites. The constructed vector was transformed to E. coli BL21 as an expression host and induced in the presence of IPTG for expression of the IGFBP3 protein. Protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS:Double digestion of the new plasmid (pET-11a -IGBP3) with NdeI and BamHI showed two bands in 873 bp and 5700 bp. To study the accurate cloning procedure, the plasmid was sequenced and its authenticity was confirmed. Also the expected protein band (31.6 kDa) was observed in SDS-PAGE analysis. CONCLUSION:DNA fragment encoding the full-length IGFBP3 protein was accurately cloned in the pET-11a expression vector and the recombinant plasmid transformed to E. coli BL21 (DE3) expression host. Results of the SDS-PAGE analysis verified that recombinant IGFBP3 (31.6 kDa) are successfully expressed under the control of T7 promoter. As we shown pET-11a can be successfully used for expression of the IGFBP3 protein.

SUBMITTER: Khodadadi E

PROVIDER: S-EPMC4386200 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli.

Khodadadi Emad E Panjepour Mojtaba M Abbasian Mahdi M Broujeni Zahra Khalili ZK Mofid Mohammad Reza MR

Advanced biomedical research 20150325

<h4>Background</h4>The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this research we cloned and expressed the full-length IGFBP3 in the pET-11a vector and BL21 (DE3) expression host.<h4>Materials and methods</h4>First the sequence encoding of IGFBP3 was designed based ...[more]

PMID: 25878991

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Project description:BackgroundInsulin-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 methodsFirst, 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).ResultsThe 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.ConclusionWe 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.

Project description:BackgroundInsulin-like growth factor 1 (IGF1) stimulates mitosis and inhibits apoptosis. Some published results have shown an association between circulating IGF1 and breast-cancer risk, but it has been unclear whether this relationship is consistent or whether it is modified by IGF binding protein 3 (IGFBP3), menopausal status, oestrogen receptor status or other factors. The relationship of IGF1 (and IGFBP3) with breast-cancer risk factors is also unclear. The Endogenous Hormones and Breast Cancer Collaborative Group was established to analyse pooled individual data from prospective studies to increase the precision of the estimated associations of endogenous hormones with breast-cancer risk.MethodsIndividual data on prediagnostic IGF1 and IGFBP3 concentrations were obtained from 17 prospective studies in 12 countries. The associations of IGF1 with risk factors for breast cancer in controls were examined by calculating geometric mean concentrations in categories of these factors. The odds ratios (ORs) with 95% CIs of breast cancer associated with increasing IGF1 concentrations were estimated by conditional logistic regression in 4790 cases and 9428 matched controls, with stratification by study, age at baseline, and date of baseline. All statistical tests were two-sided, and a p value of less than 0.05 was considered significant.FindingsIGF1 concentrations, adjusted for age, were positively associated with height and age at first pregnancy, inversely associated with age at menarche and years since menopause, and were higher in moderately overweight women and moderate alcohol consumers than in other women. The OR for breast cancer for women in the highest versus the lowest fifth of IGF1 concentration was 1.28 (95% CI 1.14-1.44; p<0.0001). This association was not altered by adjusting for IGFBP3, and did not vary significantly by menopausal status at blood collection. The ORs for a difference in IGF1 concentration between the highest and lowest fifth were 1.38 (95% CI 1.14-1.68) for oestrogen-receptor-positive tumours and 0.80 (0.57-1.13) for oestrogen-receptor-negative tumours (p for heterogeneity=0.007).InterpretationCirculating IGF1 is positively associated with breast-cancer risk. The association is not substantially modified by IGFBP3, and does not differ markedly by menopausal status, but seems to be confined to oestrogen-receptor-positive tumours.FundingCancer Research UK.

Dataset Information

Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli.

Publications

Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli.

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