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

Khodadadi, Emad; Panjepour, Mojtaba; Abbasian, Mahdi; Broujeni, Zahra Khalili; Reza Mofid, Mohammad

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

Authors

Department of Biochemistry, School of Pharmacy, Bioinformatics Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

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.

Keywords

References

1.	Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA: Cancer J Clin 2011;61:69-90.
2.	Jafari S, Babaeipour V, Seyedi H, Rahaie M, Mofid MR, Haddad L, et al. Recombinant production of mecasermin in E. coli expression system, Research in Pharmaceutical Sciences; 2014. p. 453-61.
3.	Paz K, Hadari YR. Targeted therapy of the insulin-like growth factor-1 receptor in cancer. Comb Chem High Throughput Screen 2008;11:62-9.
4.	Kemp SF. Insulin-like growth factor-I deficiency in children with growth hormone Insensitivity: Current and future treatment options. BioDrugs 2009;23:155-63.
5.	Shimasaki S, Ling N. Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5 and -6). Prog Growth Factor Res 1991;3:243-66.
6.	Delafontaine P, Song YH, Li Y. Expression, regulation, and function of IGF-1, IGF-1R, and IGF-1 binding proteins in blood vessels. Arterioscler Thromb Vasc Biol 2004;24:435-44.
7.	Fletcher O, Gibson L, Johnson N, Altmann DR, Holly JM, Ashworth A, et al. Polymorphisms and circulating levels in the insulin-like growth factor system and risk of breast cancer: A systematic review. Cancer Epidemiol Biomarkers Prev 2005;14:2-19.
8.	Davison Z, de Blacquière GE, Westley BR, May FE. Insulin-like growth factor-dependent proliferation and survival of triple-negative breast cancer cells: Implications for therapy. Neoplasia 2011;13:504-15.
9.	Yu H, Rohan T. Role of the insulin-like growth factor family in cancer development and progression. J Natl Cancer Inst 2000;92:1472-89.
10.	Bartucci M, Morelli C, Mauro L, Andò S, Surmacz E. Differential insulin-like growth factor I receptor signaling and function in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. Cancer Res 2001;61:6747-54.
11.	Zhu C, Qi X, Chen Y, Sun B, Dai Y, Gu Y. PI3K/Akt and MAPK/ERK1/2 signaling pathways are involved in IGF-1-induced VEGF-C upregulation in breast cancer. J Cancer Res Clin Oncol 2011;137:1587-94.
12.	Stolzenberg-Solomon RZ, Limburg P, Pollak M, Taylor PR, Virtamo J, Albanes D. Insulin-like growth factor (IGF)-1, IGF-binding protein-3, and pancreatic cancer in male smokers. Cancer Epidemiol Biomarkers Prev 2004;13:438-44.
13.	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.
14.	Giles ED, Singh G. Role of insulin-like growth factor binding proteins (IGFBPs) in breast cancer proliferation and metastasis. Clin Exp Metastasis 2003;20:481-7.
15.	Fowlkes JL, Serra DM. Characterization of glycosaminoglycan-binding domains present in insulin-like growth factor-binding protein-3. J Biol Chem 1996;271:14676-9.
16.	Key TJ, Appleby PN, Reeves GK, Roddam AW; Endogenous Hormones and Breast Cancer Collaborative Group. Insulin-like growth factor 1(IGF1), IGF binding protein 3 (IGFBP3), and breast cancer risk: Pooled individual data analysis of 17 prospective studies. Lancet Oncol 2010;11:530-42.
17.	Liu B, Lee HY, Weinzimer SA, Powell DR, Clifford JL, Kurie JM, et al. Direct functional interactions between insulin-like growth factor-binding protein-3 and retinoid X receptor-alpha regulate transcriptional signaling and apoptosis. J Biol Chem 2000;275:33607-13.
18.	Spratt SK, Tatsuno GP, Sommer A. Cloning and characterization of bovine insulin-like growth factor binding protein-3 (bIGFBP-3). Biochem Biophys Res Commun 1991;177:1025-32.
19.	Zareie R, Abbasian M. Method to produce recombinant MBP8298 and other polypeptides by nucleotide structure optimization. US Patent No: US8527211B2; 2013.
20.	Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. Vol. 1-3. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001. p. 1.110
21.	Ibrahim YH, Yee D. Insulin-like growth factor-I and breast cancer therapy. Clin Cancer Res 2005;11:944-50s.
22.	Huang G, Dang ZF, Dang YM, Cai W, Li Y, Chen YR, et al. Expression and underlying roles of IGFBP-3 in paclitaxel-treated gastric cancer SGC-7901 Cells. Asian Pac J Cancer Prev 2014;15:5741-45.
23.	Sørensen H, Mortensen K. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 2005;4:1.
24.	Correa A, Oppezzo P. Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: Advantages of high-throughput screening. Biotechnol J 2011;6:715-30.
25.	Hatahet F, Nguyen VD, Salo KE, Ruddock LW. Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microb Cell Fact 2010;9:67.
26.	Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. Vol. 1-3. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001. p. 3.15
27.	Wu C, Yao G, Zou M, Chen G, Wang M, Wang J, et al. High-efficient and soluble expression, purification and bioassay of IGFBP-3. Chin J Biotechnol 2007;23:398-402.

Advanced Biomedical Research