Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany.
PLoS One. 2013 Sep 2;8(9):e72925. doi: 10.1371/journal.pone.0072925. eCollection 2013.
Split intein enabled protein trans-splicing (PTS) is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. PTS activity is strongly influenced by the amino acids directly flanking the splice junctions. However, to date no reliable prediction can be made whether or not a split intein is active in a particular foreign extein context. Here we describe SPLICEFINDER, a PCR-based method, allowing fast and easy screening for active split intein insertions in any target protein. Furthermore we demonstrate the applicability of SPLICEFINDER for segmental isotopic labeling as well as for the generation of multi-domain and enzymatically active proteins.
分裂内含肽介导的蛋白质转剪接（PTS）是连接两个蛋白质片段的有力方法，从而为各种蛋白质修饰或蛋白质功能控制应用铺平了道路。PTS 活性受剪接连接点直接侧翼的氨基酸强烈影响。然而，迄今为止，还不能可靠地预测分裂内含肽在特定的外源外显子环境中是否具有活性。在这里，我们描述了 SPLICEFINDER，一种基于 PCR 的方法，允许快速、轻松地筛选任何靶蛋白中活性分裂内含肽的插入。此外，我们还证明了 SPLICEFINDER 适用于分段同位素标记以及生成多结构域和具有酶活性的蛋白质。
