Antibiotic resistant genes split profiling

Splitting a protein at a position may lead to self- or assisted complementary fragments depending on whether two resulting fragments can reconstitute to maintain the native function spontaneously or require assistance from two interacting molecules. Assisted complementary fragments with high contrast are an important tool for probing biological interactions. However, only a small number of assisted-complementary split-variants have been identified as a result of manual, labor-intensive optimization of candidate genes. Here, we introduce a technique for high-throughput split-protein profiling (HiTS) that allows fast identification of self- and assisted complementary positions by transposon mutagenesis, a rapamycin-regulated FRB-FKBP protein interaction pair, and deep sequencing. We test this technique by profiling three antibiotic-resistant genes (fosfomycin-resistant gene, fosA3, erythromycin-resistant gene, ermB, and chloramphenicol-resistant gene, catI). Self- and assisted complementary fragments discovered by the high-throughput technique were subsequently confirmed by low-throughput testing of individual split positions. Thus, the HiTS technique provides a quicker alternative for discovering the proteins with suitable self- and assistant complementary split positions

在特定位点切割蛋白质可产生自互补片段或辅助互补片段，具体类型取决于切割产生的两个片段能否自发重组装以维持蛋白质的天然功能，或是需要借助两个相互作用分子的辅助。具有高对比度的辅助互补片段，是探究生物相互作用的重要工具。然而，由于需通过人工开展耗时费力的候选基因优化工作，目前仅发现了少量辅助互补型分裂变体。本研究提出一种高通量蛋白质分裂谱分析技术（high-throughput split-protein profiling, HiTS），该技术可借助转座子诱变、雷帕霉素调控的FRB-FKBP蛋白质相互作用对以及深度测序，快速鉴定自互补与辅助互补位点。我们通过对三种抗生素抗性基因——磷霉素抗性基因fosA3、红霉素抗性基因ermB以及氯霉素抗性基因catI——进行谱分析，对该技术进行了验证。通过该高通量技术发现的自互补与辅助互补片段，后续通过对单个分裂位点的低通量实验得到了验证。因此，HiTS技术为筛选具备合适自互补与辅助互补分裂位点的蛋白质提供了一种更高效的备选方案。