Synthesis of mammalian profilin in Escherichia coli and its characterization.
Synthesis of mammalian profilin in Escherichia coli and its characterization.
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大肠杆菌中哺乳动物profilin的合成及其表征。
DOI：
10.1002/cm.970140209
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发表时间：
1989
影响因子：
--
通讯作者：
Rubenstein,PA
中科院分区：
文献类型：
--
作者：
Babcock,G;Rubenstein,PA
Profilin 是一种 G-肌动蛋白结合蛋白，可能在控制细胞内 G/F 肌动蛋白的比例方面发挥作用。为了设计一种获得大量处于活性状态的哺乳动物 profilin 的方法，我们用含有全长大鼠脾 profilin cDNA 的质粒转染大肠杆菌，该质粒与异丙基硫代半乳糖苷 (IPTG) 诱导的启动子相邻。诱导后，他们合成了一种 15,000 MW 的新蛋白质，约占细胞总蛋白质的 5%。该蛋白质与聚 L-脯氨酸琼脂糖凝胶结合，可以用 7 M 尿素洗脱，其行为与真正的 profilin 所表现出的行为相似。超声处理后，蛋白质可以以可溶形式从细菌中释放出来，然后在 Sephadex G-75 和 DEAE A-50 Sephadex 上进行层析后，Profilin 可以纯化至均质。该蛋白质以未封闭的丙氨酸开始，表明起始甲酰基和蛋氨酸残基已被去除。基于凝胶过滤分析和肌动蛋白聚合测定，重组 profilin 从鸡骨骼肌肌动蛋白中解离的特征在于 Kdof 约为 2 μM。这些结果表明可以方便地大量制备纯化的活性哺乳动物profilin。这项研究还证明了在涉及通过定点诱变改变的突变型 profilin 的结构功能研究中使用细菌合成的 profilin 的可行性。
Profilin is a G‐actin binding protein that may have a role in controlling the ratio of G/F actin within the cells To devise a way for obtaining large amounts of mammalian profilin in an active state, we transfectedEscherichia coliwith a plasmid containing a full‐length rat spleen profilin cDNA adjacent to a promoter inducible by isopropyl thiogalactoside (IPTG). Upon induction, they synthesized a new protein of 15,000 MW constituting approximately 5% of the total cell protein. This protein bound to poly‐L‐proline Sepharose and could be eluted with 7 M urea, behavior similar to that exhibited by authentic profilin. The protein could be released from the bacteria in soluble form following sonication, and the profilin could then be purified to homogeneity following chromatography on Sephadex G‐75 and DEAE A‐50 Sephadex. The protein began with an unblocked Ala, indicating that the initiating formyl and methionine residues had been removed. The dissociation of the recombinant profilin from chicken skeletal muscle actin was characterized by a Kdof approximately 2 μM based on gel filtration analysis and actin polymerization assays. These results show that purified active mammalian profilin can be made conveniently in large quantities. This study also demonstrates the feasibility of using bacterially synthesized profilin in structure‐function studies involving mutant profilins altered by site‐directed mutagenesis.