Institute of Biochemistry and Biology, University of Potsdam, Potsdam-Golm, Germany.
PLoS One. 2011 Apr 26;6(4):e18900. doi: 10.1371/journal.pone.0018900.
Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here.
在异源宿主中进行功能性研究的蛋白质表达是一项繁琐的工作。在这里，我们报告了一个用于体内和体外平行蛋白质表达的优越平台。该平台结合了高效的连接独立克隆（LIC）和通过 N 或 C 末端融合到近红外荧光蛋白（IFP）来即时检测表达的蛋白质。对于每个开放阅读框，只需生成两个 PCR 片段（使用三个 PCR 引物），并通过 LIC 插入到十个适合微生物宿主表达蛋白质的表达载体中，包括大肠杆菌、乳酸克鲁维酵母、毕赤酵母、原生动物利什曼原虫和体外转录/翻译系统。IFP 融合蛋白的积累可以通过对活细胞或直接在 SDS-PAGE 后未经额外处理的粗蛋白提取物进行红外成像来检测。我们成功地将 LIC-IFP 平台用于十种植物和真菌蛋白的体内和体外表达，包括转录因子和酶。使用 IFP 报告基因，我们还建立了简便的方法，用于可视化蛋白质-蛋白质相互作用以及在微量滴定和无凝胶格式中检测 DNA-转录因子相互作用。我们得出结论，IFP 是一种用于高通量蛋白质表达和分析的优秀报告基因，使用这里报道的设置可以很容易地扩展到许多其他表达宿主。
