Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening
Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening
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DOI：
10.1021/bc025534z
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发表时间：
2002-07-01
影响因子：
4.7
通讯作者：
Nock, S
中科院分区：
文献类型：
--
作者：
Sydor, JR;Mariano, M;Nock, S
内含肽介导的蛋白质连接是最近开发的一种方法，可以对蛋白质进行 C 末端标记。该技术需要正确折叠的内含肽突变体，该突变体与目标蛋白的 C 末端融合以产生硫酯，从而允许肽与 N 末端半胱氨酸连接 (1, 2)。在这里，我们描述了这种方法的建立，用于在变性条件下标记作为内含肽融合蛋白不溶性表达的靶蛋白。与蟾蜍分枝杆菌 gyrA 内含肽的 GFPuv 融合蛋白在大肠杆菌的包涵体中表达，并最初用作模型蛋白来验证不同重折叠条件下的内含肽切割活性。内含肽在非变性和轻微变性条件下重折叠后显示出活性。具有抗中性亲和素单链抗体的相同内含肽的构建体也以不溶形式表达。在变性条件下，在 4 M 尿素中为该单链抗体-内含肽融合蛋白建立了内含肽介导的连接，以防止融合蛋白在第一个重折叠步骤期间发生显着沉淀。在优化条件下，单链抗体被荧光肽标记，最终重折叠后用于生物芯片上的抗原筛选。该筛选程序可以确定小型化形式的抗生物素蛋白的 scFv 的结合特征。
Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.