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 ,
Abstract: (9581 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
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