Fuhrmeister Robert, Streubel Jana
Department of Plant Biotechnology, Institute of Plant Genetics, Leibniz Universität Hannover, Hanover, Germany.
Bio Protoc. 2023 Aug 5;13(15):e4733. doi: 10.21769/BioProtoc.4733.
The study of genes and their products is an essential prerequisite for fundamental research. Characterization can be achieved by analyzing mutants or overexpression lines or by studying the localization and substrate specificities of the resulting proteins. However, functional analysis of specific proteins in complex eukaryotic organisms can be challenging. To overcome this, the use of heterologous systems to express genes and analyze the resulting proteins can save time and effort. Yeast is a preferred heterologous model organism: it is easy to transform, and tools for genomics, engineering, and metabolomics are already available. Here, we describe a well-established and simple method to analyze the activity of plant monosaccharide transporters in the baker's yeast, Saccharomyces cerevisiae, using a simple growth complementation assay. We used the famous hexose-transport-deficient yeast strain EBY.VW4000 to express candidate plant monosaccharide transporters and analyzed their transport activity. This assay does not require any radioactive labeling of substrates and can be easily extended for quantitative analysis using growth curves or by analyzing the transport rates of fluorescent substrates like the glucose analog 2-NBDG. Finally, to further simplify the cloning of potential candidate transporters, we provide level 0 modular cloning (MoClo) modules for efficient and simple Golden Gate cloning. This approach provides a convenient tool for the functional analysis of plant monosaccharide transporters in yeast. Key features Comprehensive, simple protocol for analysis of plant monosaccharide transporters in yeast Includes optional MoClo parts for cloning with Golden Gate method Includes protocol for the production and transformation of competent yeast cells Does not require hazardous solutions, radiolabeled substrates, or specialized equipment.
对基因及其产物的研究是基础研究的重要前提。可以通过分析突变体或过表达系，或者通过研究所得蛋白质的定位和底物特异性来实现表征。然而，在复杂的真核生物中对特定蛋白质进行功能分析可能具有挑战性。为了克服这一问题，使用异源系统来表达基因并分析所得蛋白质可以节省时间和精力。酵母是一种首选的异源模式生物：它易于转化，并且已经有了基因组学、工程学和代谢组学的工具。在这里，我们描述了一种成熟且简单的方法，使用简单的生长互补试验来分析面包酵母（酿酒酵母）中植物单糖转运蛋白的活性。我们使用著名的缺乏己糖转运能力的酵母菌株EBY.VW4000来表达候选植物单糖转运蛋白，并分析它们的转运活性。该试验不需要对底物进行任何放射性标记，并且可以很容易地通过生长曲线或分析荧光底物（如葡萄糖类似物2-NBDG）的转运速率来扩展进行定量分析。最后，为了进一步简化潜在候选转运蛋白的克隆，我们提供了0级模块化克隆（MoClo）模块，用于高效且简单的金门克隆。这种方法为在酵母中对植物单糖转运蛋白进行功能分析提供了一个方便的工具。关键特性用于分析酵母中植物单糖转运蛋白的全面、简单方案包括用于金门法克隆的可选MoClo部件包括制备和转化感受态酵母细胞的方案不需要危险溶液、放射性标记底物或专门设备。
