Genomics, BioInnovationsZentrum, Technische Universitaet Dresden, Am Tatzberg 47, 01307 Dresden, Germany.
Methods. 2011 Feb;53(2):113-9. doi: 10.1016/j.ymeth.2010.09.003. Epub 2010 Sep 22.
Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. BAC (bacterial artificial chromosome) transgenes present a way to express a chosen protein at physiological levels with all regulatory elements in their native configurations, including cell cycle, alternative splicing and microRNA regulation. Recombineering has become the method of choice for modifying large constructs like BACs. Here, we present a method for protein tagging by recombineering BACs, transfecting cells and evaluating tagged protein expression.
蛋白标签为蛋白质组学和调控组学研究提供了许多优势，特别是由于使用了通用且高度敏感的方法，这些方法可以以合理的通量应用。理想情况下，蛋白标签等同于对每个选定的蛋白都有高亲和力的抗体。然而，如果标记的蛋白过表达，这些优势就会受到影响，这通常是 cDNA 表达载体的情况。BAC（细菌人工染色体）转基因提供了一种以生理水平表达选定蛋白的方法，所有的调控元件都处于其天然构象，包括细胞周期、选择性剪接和 microRNA 调控。同源重组已成为修饰像 BAC 这样的大型构建体的首选方法。在这里，我们提出了一种通过同源重组 BAC、转染细胞和评估标记蛋白表达来进行蛋白标记的方法。
