Heterologous Overexpression of Membrane-Anchored Subunit II of Spinach Chloroplast ATP Synthase and Its Detergent-Free Purification as a Soluble Protein
Heterologous Overexpression of Membrane-Anchored Subunit II of Spinach Chloroplast ATP Synthase and Its Detergent-Free Purification as a Soluble Protein
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DOI：
10.1515/znc-1999-3-413
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发表时间：
1999-04
期刊：
影响因子：
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通讯作者：
Hans-Jürgen Tiburzy;M. Zimmermann;R. Oworah-Nkruma;R. Berzborn
中科院分区：
文献类型：
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作者：
Hans-Jürgen Tiburzy;M. Zimmermann;R. Oworah-Nkruma;R. Berzborn
亚基II是叶绿体ₒ合酶的四个不完全相同的亚基之一。在菠菜叶绿体中，它是唯一的核编码的Cfₒ亚基。据推测，CfₒII不是光合作用生物体中典型的附加亚基，在大肠杆菌中没有对应的亚基，但相当于E.colib亚基(Tiburzy，H.-J.和BerzBorn，R.J.(1997)，Z.Naturforsch)。52C、789-798)。利用细菌表达载体Pt7-7实现了亚基II的异源表达。重组亚单位II(IIrec)既不整合到细菌膜上，也不沉淀成包涵体。凝胶过滤层析表明，IIrec形成了更高级别的聚集体。在三个层析步骤中，大约。在不使用洗涤剂的情况下，从一升细菌培养中可获得10毫克的可溶性IIrec，其电泳均一。因此，真核膜锚定蛋白在大肠杆菌中得到了高效表达，并以可溶性形式进行了纯化。
Subunit II is one of the four nonidentical subunits of the membrane integral, protontransporting moiety (CFₒ) hloroplast ATP synthase. In chloroplasts of spinach leaves, it is the only nuclear-encoded CFₒ subunit. It has been deduced that CFₒII is not an additional subunit typical for photosynthetic organisms with no counterpart in E.coli, but equivalent to E. coli subunit b (Tiburzy, H.-J. and Berzborn, R. J. (1997), Z. Naturforsch. 52c, 789-798). Heterologous expression of subunit II was achieved by using the bacterial expression vector pT7-7. Recombinant subunit II (IIrec) does not integrate into the bacterial membrane nor does it precipitate into inclusion bodies. Gel filtration chromatography indicates that IIrec forms higher order aggregates. In three chromatographic steps approx. 10 mg of soluble IIrec of electrophoretic homogeneity are obtained from one liter of bacterial culture without using detergents. Thus, a eukaryotic membrane-anchored protein has been overexpressed in E. coli and has been purified in a soluble form.