Adipose differentiation related protein: expression, purification of recombinant protein in Escherichia coli and characterization of its fatty acid binding properties.
Adipose differentiation related protein: expression, purification of recombinant protein in Escherichia coli and characterization of its fatty acid binding properties.
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DOI：
10.1016/s1388-1981(00)00128-1
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发表时间：
2000-11
期刊：
影响因子：
--
通讯作者：
G. Serrero;A. Frolov;F. Schroeder;K. Tanaka;L. Gelhaar
中科院分区：
文献类型：
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作者：
G. Serrero;A. Frolov;F. Schroeder;K. Tanaka;L. Gelhaar
脂肪分化相关蛋白（ADRP）是一种分子量为53 kDa的蛋白质，由一个最初通过差异杂交从小鼠脂肪细胞中克隆的cDNA编码。ADRP在脂肪分化程序的早期启动期间被诱导，并且在成熟脂肪细胞中以高水平表达。我们已经证明，ADRP刺激脂肪酸的摄取，从而为ADRP在脂质代谢中的功能作用提供了证据。在本文中，鼠ADRP已表达为重组组氨酸标记的蛋白在大肠杆菌中，并从表达培养物中纯化，以检查其生化特性。我们在这里报告，纯化的重组ADRP结合脂肪酸和NBD-硬脂酸表现出化学计量饱和结合的Kd=0.145±0.003 μM和Bmax=0.99±0.05。荧光发射光谱的分析表明，ADRP结合位点的极性接近于23，接近于在其他脂质结合蛋白如肝脂肪酸结合蛋白中观察到的脂肪酸结合位点。本文提供的数据提供了证据，证明在本文所述的实验条件下纯化的分离的ADRP可用于功能研究。
Adipose differentiation related protein (ADRP) is a 53 kDa protein encoded by a cDNA originally cloned by differential hybridization from murine adipocytes. ADRP is induced during the early onset of the adipose differentiation program and is expressed at high level in mature adipocytes. We have demonstrated that ADRP stimulated the uptake of fatty acids thereby providing evidence for a functional role of ADRP in lipid metabolism. In the present paper, the murine ADRP has been expressed as a recombinant histidine-tagged protein in Escherichia coli, and purified from expressing cultures in order to examine its biochemical properties. We report here that the purified recombinant ADRP binds fatty acids and exhibits stoichiometric saturable binding of NBD-stearic acid with a Kd=0.145±0.003 μM and a Bmax=0.99±0.05. Analysis of fluorescence emission spectra indicates that the polarity of the ADRP binding site is near ϵ≈23, close to that observed for fatty acid binding sites in other lipid binding proteins such as the liver fatty acid binding protein. The data presented here provide evidence that isolated ADRP purified in the experimental conditions described here can be used for functional studies.