Expression, purification, and characterization of recombinant human flotillin-1 in Escherichia coli
Expression, purification, and characterization of recombinant human flotillin-1 in Escherichia coli
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DOI：
10.1016/j.pep.2005.03.001
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发表时间：
2005-07-01
影响因子：
1.6
通讯作者：
Zhang, ZH
中科院分区：
文献类型：
--
作者：
Ding, Y;Jiang, M;Zhang, ZH
为了提高人flotillin-1（reggie-2）的表达量，克隆了人flotillin-1（reggie-2）基因，并在大肠杆菌中表达了4种不同的融合标签（hexahistidine，glutathione S-transferase，NusA和thioredoxin）。将表达量最高的带硫氧还蛋白标签的flotillin-1从包涵体中溶解出来，首先在变性条件下用固定化金属亲和柱纯化，然后用尿素梯度递减法在柱上直接复性。凝血酶裂解硫氧还蛋白标签，阴离子交换柱和凝胶过滤柱进一步纯化flotillin-1蛋白。纯化蛋白经变性凝胶电泳和Western blot鉴定。从1 L培养基中典型产量为3.4mg，纯度大于98%。通过pull-down实验，证实了重组flotillin-1和人红细胞膜天然flotillin-1与c-Cbl-associated protein或neuroglobin的相互作用，表明重组蛋白具有功能活性。这是第一次报道了大量高纯度的活性重组筏特异性蛋白的表达、纯化和表征，这将有助于进一步的研究，如X射线晶体学。(c)2005年爱思唯尔公司All rights reserved.
Human flotillin-1 (reggie-2), a major hydrophobic protein of biomembrane microdomain lipid rafts, was cloned and expressed in Escherichia coli with four different fusion tags (hexahistidine, glutathione S-transferase, NusA, and thioredoxin) to increase the yield. The best expressed flotillin-1 with thioredoxin tag was solubilized from inclusion bodies, first purified by immobilized metal affinity column under denaturing condition and direct refolded on column by decreasing urea gradient method. The thioredoxin tag was cleaved by thrombin, and the flotillin-1 protein was further purified by anion exchanger and gel filtration column. The purified protein was verified by denaturing gel electrophoresis and Western blot. The typical yield was 3.4 mg with purity above 98% from 1 L culture medium. Using pull-down assay, the interaction of both the recombinant flotillin-1 and the native flotillin-1 from human erythrocyte membranes with c-Cbl-associated protein or neuroglobin was confirmed, which demonstrated that the recombinant proteins were functional active. This is the first report describing expression, purification, and characterization of active recombinant raft specific protein in large quantity and highly purity, which would facilitate further research such as X-ray crystallography. (c) 2005 Elsevier Inc. All rights reserved.