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首页> 外文期刊>Protein Expression and Purification >Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein
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Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein

机译：双功能解整合蛋白/碱性磷酸酶杂合蛋白的克隆，表达和表征

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摘要

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alpha(llb)beta(3) integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alpha(llb)beta(3) integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alpha(llb)beta(3) integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alpha(llb)beta(3) integrin is involved. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 25]
机译：整联蛋白是负责细胞通讯的跨膜异二聚糖蛋白。因此，它们在许多生理事件中起着至关重要的作用。毒蛇蛇毒中含有被称为双整合素的整合素拮抗剂，它们可以结合并抑制整合素的功能。他们提出了一个包含负责整合素结合的RGD图案的环。与报告酶融合的整合素的工程改造将是构建整合素标记的有趣方法。甚至更多，双整合蛋白支架可用于呈现其他蛋白质结合基序。在这项工作中，我们已经通过克隆表皮中的依斯托他汀DNA并将其表达到pLIP6-GN载体中而获得了碱性磷酸酶（APv）标记的表皮他汀（Er）。 Eristostatin，一个49个残基的整合素，选择性地与alpha（11b）beta（3）整合素结合，抑制了它与纤维蛋白原的结合。通过SDS-PAGE和使用抗Er和抗AP抗体的Western印迹鉴定得到的融合蛋白Er / APv。该融合蛋白显示出与野生型APv相似的酶促AP活性，并且使用固定化细胞与标记物孵育并通过AP底物进行了一步印迹分析，测试了其在alpha（llb）beta（3）整联蛋白测定中的潜在用途。 Er / APv对血小板和alpha（IIb）beta（3）整合素转染的细胞表现出选择性，并与未标记的Er在同一区域发生反应，如在竞争分析中所分析。我们的数据提供了一种新颖的工具Er / APv，可在涉及alpha（llb）beta（3）整合素的过程中用作分子标记。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：25]

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