Volume 9, Issue 2 (2018)
JMBS 2018, 9(2): 165-169 |
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Nakhaei Amroudie M, Ataei F. Expression, Purification, and Characterization of IP3-binding Domain from Human Type 2. JMBS 2018; 9 (2) :165-169
URL: http://biot.modares.ac.ir/article-22-24322-en.html
URL: http://biot.modares.ac.ir/article-22-24322-en.html
1- Biochemistry Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran
2- Biochemistry Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran, Biochemistry Department, Biological Sciences Faculty, Tarbiat Modares University, Nasr Bridge, Jalal-Al-Ahmad Highway, Tehran, Iran , Ataei_f@modares.ac.ir
2- Biochemistry Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran, Biochemistry Department, Biological Sciences Faculty, Tarbiat Modares University, Nasr Bridge, Jalal-Al-Ahmad Highway, Tehran, Iran , Ataei_f@modares.ac.ir
Abstract: (8530 Views)
Aims: IP3 is a key regulator molecule in the message transmission pathway, and releases calcium into the cytoplasm by binding intracellular IP3R receptors on the surface of the internal calcium stores. The aim of this study was expression, purification, and characterization of IP3-binding domain from human type 2.
Materials and Methods: In this experimental study, the pET-28a plasmid of the carrier of the IP3BD gene was transferred to the E.coli expression strain BL21 (DE3) by chemical method. In order to optimize the expression in the bacterial system, the expression was studied in different conditions, and various temperatures such as 16, 18, 20, and 24°C, the different times after incubation, type of inducer, and its different concentrations were investigated. The induced bacteria were purified on the basis of thermal shock through nickel column for chromatography and the purity of the protein was measured through SDS-PAGE. The fluorescence emission of IP3-binding domain was measured in the presence and absence of an IP3 ligand at wavelength of 295nm.
Findings: Protein did not have a significant expression in LB, TB, and 2xYT environments, and no changes were observed at different times. Expression of bacterial protein at 20°C based on thermal shock of 42°C was higher than in all cases. The purification of the induced bacteria was difficultly repeated due to thermal shock, and the purified samples did not have high concentrations. The fluorescence emission of the protein decreased in the presence of the IP3 ligand.
Conclusion: The bacterial expression of IP3-binding domain from human type 2 is weak, but the expression of protein increases with the induction of shock of 42°C.
Materials and Methods: In this experimental study, the pET-28a plasmid of the carrier of the IP3BD gene was transferred to the E.coli expression strain BL21 (DE3) by chemical method. In order to optimize the expression in the bacterial system, the expression was studied in different conditions, and various temperatures such as 16, 18, 20, and 24°C, the different times after incubation, type of inducer, and its different concentrations were investigated. The induced bacteria were purified on the basis of thermal shock through nickel column for chromatography and the purity of the protein was measured through SDS-PAGE. The fluorescence emission of IP3-binding domain was measured in the presence and absence of an IP3 ligand at wavelength of 295nm.
Findings: Protein did not have a significant expression in LB, TB, and 2xYT environments, and no changes were observed at different times. Expression of bacterial protein at 20°C based on thermal shock of 42°C was higher than in all cases. The purification of the induced bacteria was difficultly repeated due to thermal shock, and the purified samples did not have high concentrations. The fluorescence emission of the protein decreased in the presence of the IP3 ligand.
Conclusion: The bacterial expression of IP3-binding domain from human type 2 is weak, but the expression of protein increases with the induction of shock of 42°C.
Subject:
Agricultural Biotechnology
Received: 2016/10/19 | Accepted: 2017/12/5 | Published: 2018/06/21
Received: 2016/10/19 | Accepted: 2017/12/5 | Published: 2018/06/21
References
1. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature. 1993;361(6410):315-25. [Link] [DOI:10.1038/361315a0]
2. Berridge MJ. Cell signalling. A tale of two messengers. Nature. 1993;365(6445):388-9. [Link] [DOI:10.1038/365388a0]
3. Maeda N, Niinobe M, Mikoshiba K. A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex. EMBO J. 1990;9(1):61-7. [Link] [DOI:10.1002/j.1460-2075.1990.tb08080.x]
4. Koga T, Yoshida Y, Cai JQ, Islam MO, Imai S. Purification and characterization of 240-kDa cGMP-dependent protein kinase substrate of vascular smooth muscle. Close resemblance to inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1994;269(15):11640-7. [Link]
5. O'Rourke F, Matthews E, Feinstein MB. Purification and characterization of the human type 1 Ins(1,4,5)P3 receptor from platelets and comparison with receptor subtypes in other normal and transformed blood cells. Biochem J. 1995;312(Pt 2):499-503. [Link] [DOI:10.1042/bj3120499]
6. Mignery GA, Newton CL, Archer BT 3rd, Südhof TC. Structure and expression of the rat inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1990;265(21):12679-83. [Link]
7. Yamamoto-Hino M, Sugiyama T, Hikichi K, Mattei MG, Hasegawa K, Sekine S, et al. Cloning and characterization of human type 2 and type 3 inositol 1,4,5-trisphosphate receptors. Receptors channels. 1994;2(1):9-22. [Link]
8. Maranto AR. Primary structure, ligand binding, and localization of the human type 3 inositol 1,4,5-trisphosphate receptor expressed in intestinal epithelium. J Biol Chem. 1994;269(2):1222-30. [Link]
9. De Smedt H, Missiaen L, Parys JB, Bootman MD, Mertens L, Van Den Bosch L, et al. Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. The J biol chem. 1994;269(34):21691-8. [Link]
10. Taylor CW, Da Fonseca PCA, Morris EP. IP3 receptors: The search for structure. Trends biochem sci. 2004;29(4):210-9. [Link] [DOI:10.1016/j.tibs.2004.02.010]
11. Furuichi T, Simon-Chazottes D, Fujino I, Yamada N, Hasegawa M, Miyawaki A, et al. Widespread expression of inositol 1,4,5-trisphosphate receptor type 1 gene (Insp3r 1) in the mouse central nervous system. Receptors channels. 1993;1(1):11-24. [Link]
12. Yoshida Y, Imai S. Structure and function of inositol 1,4,5-trisphosphate receptor. Jpn J pharmacol. 1997;74(2):125-37. [Link] [DOI:10.1254/jjp.74.125]
13. Bosanac I, Alattia JR, Mal TK, Chan J, Talarico S, Tong FK, et al. Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand. Nature. 2000;420(6916):696-700. [Link] [DOI:10.1038/nature01268]
14. Ataei F, Torkzadeh-Mahani M, Hosseinkhani S. A novel luminescent biosensor for rapid monitoring of IP3 by split-luciferase complementary assay. Biosens Bioelectron. 2013;41:642-8. [Link] [DOI:10.1016/j.bios.2012.09.037]
15. Arsène F, Tomoyasu T, Bukau B. The heat shock response of Escherichia coli. Int J Food Microbiol. 2000;55(1-3):3-9. [Link] [DOI:10.1016/S0168-1605(00)00206-3]
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