Production and Characterization of Chimeric Transferrins for the Determination of the Binding Domains for Bacterial Transferrin Receptors (*)
Production and Characterization of Chimeric Transferrins for the Determination of the Binding Domains for Bacterial Transferrin Receptors (*)
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DOI：
10.1074/jbc.271.2.1166
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发表时间：
1996-01
期刊：
影响因子：
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通讯作者：
M. Retzer;A. Kabani;L. L. Button-L.;R. Yu;A. Schryvers
中科院分区：
文献类型：
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作者：
M. Retzer;A. Kabani;L. L. Button-L.;R. Yu;A. Schryvers
NeisSeraceae和Pasteurellaceae中的病原菌具有与宿主转铁蛋白特异性结合的外膜蛋白，这是铁获取过程的第一步。作为先前使用生化方法研究配体-受体相互作用的合乎逻辑的进展，我们启动了一种涉及产生重组嵌合转铁蛋白的方法，以进一步确定参与受体结合的转铁蛋白区域。为了制备牛/人杂交体，克隆了牛转铁蛋白基因，并对其进行了测序，并与已有的人转铁蛋白基因序列进行了比较。在确定了潜在的剪接位点后，利用基于聚合酶链式反应的重叠延伸剪接方法构建了杂交转铁蛋白基因。构建了5个包含牛和人转铁蛋白序列的杂交基因。重组转铁蛋白在杆状病毒表达载体系统中表达，并用刀豆蛋白A-琼脂糖亲和纯化。通过固相结合实验和亲和分离实验，分析重组蛋白与多克隆抗体和单抗的反应性，以及与脑膜炎奈瑟氏菌转铁蛋白受体蛋白的结合。这些实验使我们能够定位人类转铁蛋白主要参与与脑膜炎奈瑟氏菌受体结合的氨基酸残基346-588的区域。这些嵌合体的构建为研究转铁蛋白与人和牛的细菌病原体受体的结合提供了独特的工具。
Pathogenic bacteria in the Neisseriaceae and Pasteurellaceae possess outer membrane proteins that specifically bind transferrin from the host as the first step in the iron acquisition process. As a logical progression from prior studies of the ligand-receptor interaction using biochemical approaches, we have initiated an approach involving the production of recombinant chimeric transferrins to further identify the regions of transferrin involved in receptor binding. In order to prepare bovine/human hybrids, the bovine transferrin gene was cloned, sequenced, and compared with the existing human transferrin gene sequence. After identification of potential splice sites, hybrid transferrin genes were constructed using the polymerase chain reaction-based approach of splicing by overlap extension. Five hybrid genes containing sequences from both bovine and human transferrin were constructed. Recombinant transferrins were produced in a baculovirus expression vector system and affinity-purified using concanavalin A-Sepharose. The recombinant proteins were analyzed for reactivity against polyclonal and monoclonal antibodies and assessed for binding to Neisseria meningitidis transferrin receptor proteins in solid-phase binding assays and affinity isolation experiments. These experiments enabled us to localize the regions of human transferrin predominantly involved in binding to the N. meningitidis receptor to amino acid residues 346-588. The construction of these chimeras provides unique tools for the investigation of transferrin binding to receptors from both human and bovine bacterial pathogens.