Optimizing DsRed

描述（由申请人提供）： 本提案的目标是优化Discosoma红色荧光蛋白DsRed用作报告蛋白和融合标签。我们之前的工作已经产生了快速成熟的DsRed变体。目前的挑战是DsRed形成稳定的四聚体，因此，许多DsRed融合蛋白是无功能的，甚至是有毒的。因此，我们将尝试生成单体DsRed。该项目有两个具体目标。 具体目标#1：开发DsRed的单体变体。为了削弱四聚化，我们将突变两个四聚化界面处的关键残基。四聚体的进一步破坏将通过遗传筛选来完成。将通过酵母中的融合蛋白产生、非变性SDS-PAGE和分析性超离心来评估所得DsRed变体的寡聚状态。虽然最初试图消除四聚体消除荧光，我们已经获得了新的DsRed变体，即使在两个四聚体界面的诱变后也会产生强荧光。这些新的变体应该为创建DsRed单体铺平道路。 具体目标#2：优化单体DsRed的光谱特性。单体DsRed变体仅在其保留明亮的红色荧光和最小的绿色发射时才有用。将DsRed转化为单体可以减少红色荧光，并且可能通过阻止成熟DsRed中存在的红色和绿色物质之间的FRET来增强绿色发射。我们将通过单体DsRed的随机诱变来解决这些缺陷。预期的最终结果是一个DsRed变体，它将像GFP一样通用。 荧光蛋白已成为基础和应用生物医学研究的关键实验工具。这里描述的研究将导致这项技术的新应用。

DESCRIPTION (provided by applicant): The goal of this proposal is to optimize the Discosoma red fluorescent protein DsRed for use as a reporter protein and fusion tag. Our previous work has generated rapidly maturing variants of DsRed. The current challenge is that DsRed forms a stable tetramer, and as a result, many DsRed fusion proteins are nonfunctional or even toxic. We will therefore attempt to generate a monomeric DsRed. This project has two Specific Aims. Specific Aim #1: To develop a monomeric variant of DsRed. To weaken the tetramerization, we will mutate key residues at the two tetramerization interfaces. Further disruption of the tetramer will be accomplished through genetic screens. The oligomeric status of the resulting DsRed variants will be assessed by fusion protein production in yeast, nondenaturing SDS-PAGE, and analytical ultracentrifugation. Although initial attempts to eliminate the tetramerization abolished the fluorescence, we have obtained new DsRed variants that develop strong fluorescence even after mutagenesis of both tetramerization interfaces. These new variants should pave the way for the creation of a DsRed monomer. Specific Aim #2: To optimize the spectral properties of monomeric DsRed. A monomeric DsRed variant will only be useful if it retains bright red fluorescence with minimal green emission. Converting DsRed to a monomer may diminish the red fluorescence, and is likely to boost the green emission by preventing FRET between the red and green species that are present in mature DsRed. We will address these deficiencies by random mutagenesis of monomeric DsRed. The anticipated end result is a DsRed variant that will be as versatile as GFP. Fluorescent proteins have become key experimental tools for basic and applied biomedical research. The studies described here will lead to new applications of this technology.