Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence.
Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence.
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DOI：
10.1016/j.pep.2013.03.013
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发表时间：
2013-06
影响因子：
1.6
通讯作者：
Fox, Brian G.
中科院分区：
文献类型：
--
作者：
Gromek, Katarzyna A.;Meddaugh, Hannah R.;Wrobel, Russell L.;Suchy, Fabian P.;Bingman, Craig A.;Primm, John G.;Fox, Brian G.
关键词：
σ 1受体（Sigma 1 receptor，S1 R）是一种真核细胞膜蛋白，具有细胞器间信号转导调节和分子伴侣的功能。在这里，我们报告了一个改进的表达S1 R在大肠杆菌中的融合麦芽糖结合蛋白（MBP）和高产纯化。在MBP和S1 R之间产生具有由0至5个丙氨酸残基组成的连接氨基酸序列的变体，并在几个E. coli表达菌株，以确定用于生产活性MBP-S1 R的构建体和宿主的最佳组合。在接头变异中，含有4-Ala接头的蛋白质表现出上级表达特性（MBP-4A-S1 R）;该构建体与大肠杆菌B834-pRARE 2和化学成分确定的生长和表达培养基配对最有效。建立了一种3步纯化方法，包括从大肠杆菌中提取;大肠杆菌膜组分使用Triton X-100和正十二烷基-β-D-吡喃麦芽糖苷的混合物，通过结合功能保留的筛选限制鉴定，并通过直链淀粉亲和和凝胶过滤色谱纯化。该程序产生约3.5mg纯化的融合蛋白/L细菌培养基。纯化的MBP-4A-S1 R在特异性配体结合活性方面显示出从起始细胞裂解物纯化175倍，并且在浓缩和冻融循环期间稳定。
Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0 to 5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with Escherichia coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ~3.5 mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling.
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DOI：
10.1126/science.1166127
发表时间：
2009-02-13
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Fontanilla D;Johannessen M;Hajipour AR;Cozzi NV;Jackson MB;Ruoho AE
通讯作者：
Ruoho AE
影响因子：
8
作者：
Center, RJ;Kobe, B;Poumbourios, P
通讯作者：
Poumbourios, P
DOI：
10.1073/pnas.96.8.4319
发表时间：
1999-04-13
影响因子：
11.1
作者：
Kobe, B;Center, RJ;Poumbourios, P
通讯作者：
Poumbourios, P
DOI：
10.1006/bbrc.1996.1842
发表时间：
1996-12-13
影响因子：
3.1
作者：
Kekuda, R;Prasad, PD;Ganapathy, V
通讯作者：
Ganapathy, V
DOI：
10.1073/pnas.93.15.8072
发表时间：
1996-07-23
影响因子：
11.1
作者：
Hanner, M;Moebius, FF;Glossmann, H
通讯作者：
Glossmann, H