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首页> 外文期刊>Protein Expression and Purification >Expression and purification of the active variant of recombinant murine Golli-interacting protein (GIP)-characterization of its phosphatase activity and interaction with Golli-BG21
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Expression and purification of the active variant of recombinant murine Golli-interacting protein (GIP)-characterization of its phosphatase activity and interaction with Golli-BG21

机译:重组鼠Golli相互作用蛋白(GIP)活性变体的表达和纯化-表征其磷酸酶活性以及与Golli-BG21的相互作用

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摘要

We have successfully expressed an active variant of recombinant murine GIP (rmGIP) with the N-terminal domain deletion (Delta N-rmGIP) in E. coli Rosetta(DE3)-RIPL cells. Whereas Delta N-rmGIP could be purified under native conditions, the purification of full-length rmGIP required denaturing conditions; and the yields were 31.4 mg and 7.4 mg per L of culture, respectively. Purity was at least 97% as assessed by HPLC. Both proteins exhibited a well-defined secondary structure composition as determined by circular dichroism spectroscopy, with a slightly higher ratio of helical and strand components in Delta N-rmGIP. The phosphatase activity of both proteins was Mg2+-dependent, with a pK(Mg) of activation being similar to 2.8 and non-cooperative binding. The Golli-myelin basic protein isoform rmBG21 (recombinant murine form) enhanced the phosphatase activity of Delta N-rmGIP below 6 mu M, but significantly inhibited it at higher concentrations. Using glutaraldehyde cross-linking and gel shift assays, the rmBG21-Delta N-rmGIP interaction was shown to be equimolar and specific, but seemingly relatively weak, suggesting that a third interaction partner is required in vivo. (C) 2008 Elsevier Inc. All rights reserved.
机译:我们已经成功地在大肠杆菌Rosetta(DE3)-RIPL细胞中表达了具有N末端域缺失(Delta N-rmGIP)的重组鼠GIP(rmGIP)的活性变体。 Delta N-rmGIP可以在天然条件下纯化,而全长rmGIP的纯化则需要变性条件。每升培养物的产量分别为31.4 mg和7.4 mg。通过HPLC评估纯度为至少97%。两种蛋白质均表现出良好的二级结构组成,这是通过圆二色光谱法确定的,在Delta N-rmGIP中螺旋和链成分的比例略高。两种蛋白质的磷酸酶活性都是Mg2 +依赖性的,激活的pK(Mg)类似于2.8和非合作结合。 Golli-髓鞘碱性蛋白同工型rmBG21(重组鼠型)在低于6μM的情况下增强了Delta N-rmGIP的磷酸酶活性,但在更高的浓度下却显着抑制了它。使用戊二醛交联和凝胶位移分析法,显示rmBG21-Delta N-rmGIP相互作用是等摩尔的和特异性的,但看似相对较弱,表明体内需要第三个相互作用伴侣。 (C)2008 Elsevier Inc.保留所有权利。

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