High efficient expression of the functional ligand binding site of the inositol 1,4,5-trisphosphate receptor in Escherichia coli
High efficient expression of the functional ligand binding site of the inositol 1,4,5-trisphosphate receptor in Escherichia coli
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DOI：
10.1006/bbrc.1999.0498
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发表时间：
1999-04-21
影响因子：
3.1
通讯作者：
Mikoshiba, K
中科院分区：
文献类型：
--
作者：
Yoshikawa, F;Uchiyama, T;Mikoshiba, K
1型肌醇1，4，5-三磷酸受体（IP（3）R1）是一种肌醇1，4，5-三磷酸（IP 3）门控的Ca 2+释放通道，在N-末端配体结合区结合IP 3。在这里，我们报告了一个改进的大肠杆菌表达系统，其中大量的IP 3结合位点可以有效地产生可溶性活性蛋白。我们已经发现，在E.大肠杆菌显著影响它们作为可溶性蛋白的产量。残基1-604（T604）含有推定的蛋白质折叠单元，产生约4.6%的总可溶性级分。结果，可溶性活性T604将为每升培养物19 mg。T604对IP 3的亲和力（Kd = 45 nM）与天然IP（3）R1的亲和力相当，而R441 Q突变体的亲和力高得多（8.1 nM）。该系统将为揭示IP（3）R1对IP 3的分子识别提供一种非常有价值和强有力的手段。(C)北京：科学出版社.
Type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1), an inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IP3 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IP3 binding constructs expressed in E. coli significantly affect their production as soluble protein. Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IP3 of T604 (K-d = 45 nM) is comparable to that of the native IP(3)R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IP(3)R1 for IP3. (C) 1999 Academic Press.