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Green fluorescent protein in Saccharomyces cerevisiae: Real-time studies of the GAL1 promoter, fusion protein expression and localization/secretion.

机译：酿酒酵母中的绿色荧光蛋白：GAL1启动子，融合蛋白表达和定位/分泌的实时研究。

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摘要

Since its cloning in 1992, Green Fluorescent Protein (GFP) has made a revolutionary impact in a wide range of fields. While majority of its applications are found in biology and/or biochemistry areas, the research goal in this work is to use GFP for bioprocess monitoring and control. This work falls into two major parts: application of GFP as a reporter to study the industrially important GAL1 promoter, and studies of GFP as a fusion tag for protein expression, localization and secretion. The yeast Saccharomyces cerevisiae was chosen as a model system.; In the first part of the project, our goal was to establish GFP's role as a real-time, on-line reporter and apply it to study various factors that might affect gene expression under control of the GAL1 promoter. The first step was to establish the correlation between GFP's fluorescence intensity measured by off-line spectrophotometer and the actual GFP concentration measured by western blot assay. In order to use GFP also as an on-line reporter, a correlation was established between GFP's off-line fluorescence intensity and on-line measurement, which was carried out by a GFP sensor developed in our lab. These studies demonstrated that GFP could indeed be used as an on-line and real-time reporter in S. cerevisiae. The tool was then applied to study the GAL1 promoter itself. Various factors including over-expression of the transcriptional activator GAL4 protein, different induction times, and especially the wide-range of inducer (galactose) amounts were examined and their impact on GFP expression studied. Then, based on the experimental results obtained, an analytical model was developed to describe the GFP expression kinetics under control of the GAL1 promoter.; In the second part of the project, our goal was to further examine GFP's role as a fusion tag and demonstrate its application for bioprocess integration. We also attempted to secrete GFP and a GFP-fusion protein with four different secretion signal peptides. We chose Hexokinase (HXK) as a model protein of interest and designed the multiple fusion HXK-EK-GFP-(His)6, where (His)6 denotes the 6x polyhistidine tag for fusion protein purification, and EK denotes the enterokinase cleavage site for fusion separation after purification. GFP's role and properties as a fusion tag was then studied in the fusion protein purification, expression, degradation, localization and secretion. A linear correlation between GFP's fluorescence intensity and HXK's enzymatic activity was obtained, indicating that GFP could be used to monitor production of its fusion partner. Confocal microscopy studies showed that GFP-HXK fusion correctly followed localization of HXK to both the cytosol and the nucleus. However, secretion of GFP or HXK-GFP to the culture medium was minimal. Expression level and localization patterns among the constructs were compared. With its monitoring capability, GFP provides the powerful possibility of following the entire bioprocess visually.
机译：自1992年克隆以来，绿色荧光蛋白（GFP）在广泛的领域中产生了革命性的影响。尽管其大多数应用是在生物学和/或生物化学领域中发现的，但这项工作的研究目标是将GFP用于生物过程的监测和控制。这项工作分为两个主要部分：使用GFP作为报告子来研究工业上重要的 GAL1 启动子，以及研究GFP作为蛋白表达，定位和分泌的融合标签。选择酵母 Saccharomyces cerevisiae 作为模型系统。在项目的第一部分中，我们的目标是确定GFP作为实时在线报告者的作用，并将其应用于研究可能影响 GAL1 启动子控制下的基因表达的各种因素。 。第一步是建立通过离线分光光度计测量的GFP荧光强度与通过蛋白质印迹测定法测量的实际GFP浓度之间的相关性。为了也将GFP用作在线报告基因，在GFP的离线荧光强度和在线测量之间建立了相关性，这是由我们实验室开发的GFP传感器进行的。这些研究表明，GFP确实可以用作 S中的在线实时报告者。啤酒。然后将该工具用于研究 GAL1 启动子本身。研究了多种因素，包括转录激活因子GAL4蛋白的过表达，不同的诱导时间，尤其是广泛的诱导剂（半乳糖）量，并研究了它们对GFP表达的影响。然后，基于获得的实验结果，建立了一个分析模型来描述在 GAL1 启动子控制下的GFP表达动力学。在项目的第二部分，我们的目标是进一步检查GFP作为融合标签的作用，并证明其在生物过程整合中的应用。我们还尝试用四个不同的分泌信号肽分泌GFP和GFP融合蛋白。我们选择己糖激酶（HXK）作为目标模型蛋白，并设计了多重融合HXK-EK-GFP-（His）6，其中（His）6代表用于融合蛋白纯化的6x多组氨酸标签，而EK代表肠激酶切割位点纯化后用于融合分离。然后在融合蛋白的纯化，表达，降解，定位和分泌中研究了GFP作为融合标签的作用和特性。获得了GFP的荧光强度和HXK的酶活性之间的线性相关性，表明GFP可以用于监测其融合伴侣的产生。共聚焦显微镜研究表明，GFP-HXK融合正确地跟随了HXK在细胞质和细胞核中的定位。但是，GFP或HXK-GFP向培养基的分泌是最小的。比较了构建体之间的表达水平和定位模式。凭借其监视功能，GFP提供了可视化跟踪整个生物过程的强大可能性。

著录项

  • 作者

    Li, Jincai.;

  • 作者单位

    University of Maryland Baltimore County.;

  • 授予单位 University of Maryland Baltimore County.;
  • 学科 Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2001
  • 页码 199 p.
  • 总页数 199
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 化工过程（物理过程及物理化学过程）;
  • 关键词

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