Manipulation of carrier proteins in antibiotic biosynthesis
Manipulation of carrier proteins in antibiotic biosynthesis
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DOI：
10.1016/j.chembiol.2004.02.010
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发表时间：
2004-02-01
影响因子：
--
通讯作者：
Burkart, MD
中科院分区：
文献类型：
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作者：
La Clair, JJ;Foley, TL;Burkart, MD
将生物合成途径工程化到合适的宿主生物体中已成为设计、评估和生产小分子疗法的一个有吸引力的场所。聚酮化合物 (PK) 和非核糖体肽 (NRP) 合酶因其模块化结构而受到特别关注，但这些酶的常规克隆和表达仍然具有挑战性。在这里，我们描述了一种利用 4'-磷酸泛酰胆碱基转移酶酶促转移修饰的辅酶 A 类似物来共价标记 PK 和 NRP 合酶载体蛋白的方法。使用这种方法，载体蛋白可以装载单一荧光或亲和报告基因，为蛋白质可视化、蛋白质印迹鉴定和亲和纯化提供新的入口。这些方法的应用提供了跟踪和量化代谢工程途径的理想工具。这些技术对于测量异源系统中的蛋白质表达、溶解度、活性和天然翻译后修饰事件很有价值。
Engineering biosynthetic pathways into suitable host organisms has become an attractive venue for the design, evaluation, and production of small molecule therapeutics. Polyketide (PK) and nonribosomal peptide (NRP) synthases have been of particular interest due to their modular structure, yet routine cloning and expression of these enzymes remains challenging. Here we describe a method to covalently label carrier proteins from PK and NRP synthases using the enzymatic transfer of a modified coenzyme A analog by a 4'-phosphopantetheinyltransferase. Using this method, carrier proteins can be loaded with single fluorescent or affinity reporters, providing novel entry for protein visualization, Western blot identification, and affinity purification. Application of these methods provides an ideal tool to track and quantify metabolically engineered pathways. Such techniques are valuable to measure protein expression, solubility, activity, and native posttranslational modification events in heterologous systems.