SUMO fusion technology for eukaryotic protein expression systems

真核蛋白表达系统的 SUMO 融合技术

• 批准号: 7481930
• 负责人: James Strickler
• 金额: $ 33.43万
• 依托单位: LIFESENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7481930
• 关键词: Adopted; Affinity; Apoptosis; Bacteria; Biological Products; Cell Cycle; Cell physiology; Cells; Cleaved cell; Endopeptidases; Engineering; Enzymes; Eukaryotic Cell; Event; Family member; Goals; Human; In Vitro; Insecta; Kinetics; Mammalian Cell; Methods; Modeling; Modification; Mutate; N-terminal; Nuclear; Organism; Peptide Hydrolases; Phase; Post-Translational Protein Processing; Process; Production; Protein Structure Initiative; Proteins; Public Health; Research; Small Ubiquitin-Related Modifier Proteins; Solubility; Stress; System; Technology; Ubiquitin; Ubiquitin Like Proteins; Ubiquitin family; Ubiquitination; United States National Institutes of Health; Variant; Work; Yeasts; base; cost; desire; improved; in vivo; interest; mutant; protein activation; protein degradation; protein expression; response

DESCRIPTION (provided by applicant): A major bottleneck in protein expression and purification is the costs associated with functional protein production in biomedical, biopharmaceutical and other research fields. Because proteins of interest originating from eukaryotic organisms often require specific components for post- translational events to mature into active forms, their functional production is crucially dependent on eukaryotic expression systems. However, most of these systems express eukaryotic proteins of interest only at relatively low yields and thus do not deliver sufficient amounts of active proteins to satisfy academic and industrial demands at reasonable costs. Bacterial hosts express proteins in enhanced amounts if the proteins are genetically fused to various protein tags, including ubiquitin family members (including the ubiquitin-like protein SUMO). A generally reliable fusion technology for quantitatively and qualitatively enhanced heterologous protein expression similar to fusions for bacterial expression has not yet been available for use in eukaryotic cells. LifeSensors' SUMO fusion technology, which is highly successful in bacterial hosts, is not yet applicable to eukaryotic cells, since these cells contain native SUMO proteases that cleave SUMO from its fusion partner in vivo. LifeSensors proposes to develop a SUMO fusion technology for functional protein production and purification in eukaryotic cells in a two step Phase I. First, a human SUMO3 fusion tag will be engineered that is not cleavable in eukaryotic host cells by mutagenizing the interaction domain of the SUMO variant with human deSUMOylase SENP2; next, a SUMO protease that can cleave the mutant SUMO tag in vitro to release the expressed protein will be engineered. Thus, protein expressed in enhanced yield but uncleaved in the host cell can be purified from the cells as a tagged fusion and subsequently cleaved to give pure protein. PUBLIC HEALTH RELEVANCE: A major bottleneck in protein expression and purification is the costs associated with functional protein production in biomedical, biopharmaceutical and other research fields. Increasing the quantity by several-fold would alleviate this problem considerably. This can be accomplished in bacteria by expressing the protein as a fusion, that is, a protein of interest joined to another protein (called SUMO). The fusion can be isolated from bacterial cells and then the SUMO part can be removed by an enzyme (protease) that recognizes SUMO. Many proteins need to be expressed in mammalian cells, unfortunately, because bacteria do not have the machinery to modify the expressed proteins and make them active. The SUMO method developed for bacteria does not work, however, for proteins expressed in eukaryotic cells, because, unlike bacteria, these cells contain enzymes that remove SUMO from the fusion before the fusion can be purified away from the other cellular proteins. LifeSensors, which developed the SUMO system for bacteria, proposes to engineer specially mutated versions of 1)SUMO that cannot be cut inside the cell by native enzymes, and 2) a mutated protease enzyme that can cut the mutated SUMO outside the cell when the fusion is purified. This combination will allow the same kind of SUMO-enhanced protein production as that afforded by bacterial systems.

描述（由申请人提供）：蛋白质表达和纯化的主要瓶颈是与生物医学，生物药物和其他研究领域中功能性蛋白质产生相关的成本。由于源自真核生物的感兴趣的蛋白质通常需要特定的成分来使翻译后事件成熟成活性形式，因此它们的功能生产至关重要地取决于真核表达系统。但是，这些系统中的大多数仅以相对较低的收益率表达感兴趣的真核蛋白，因此没有提供足够数量的活性蛋白质，以合理的成本满足学术和工业需求。如果蛋白质将蛋白质融合到各种蛋白质标签，包括泛素家族成员（包括泛素样蛋白Sumo），则细菌宿主以增强量的蛋白质表达蛋白质。一种通常可靠的融合技术，用于定量和质量增强的异源蛋白表达，类似于融合细菌表达的融合蛋白表达尚未用于真核细胞中。在细菌宿主中非常成功的救生员SUMO融合技术尚不适用于真核细胞，因为这些细胞包含天然的Sumo蛋白酶，这些蛋白酶与体内的融合伴侣裂解了Sumo。救生员建议在两步阶段的真核细胞中开发一种用于在真核细胞中的功能性蛋白质的融合技术。首先，将设计一个人类的SUMO3融合标签，在真核宿主细胞中无法通过与人desumoylase senp2的Sumo变体的相互作用域进行诱变，从而在真核宿主细胞中设计出来。接下来，将在体外裂开突变体标签以释放表达蛋白质的蛋白质。因此，以增强的产量表达但在宿主细胞中未溶解的蛋白质可以从细胞中纯化为标记的融合，然后裂解以产生纯蛋白质。公共卫生相关性：蛋白质表达和纯化的主要瓶颈是与生物医学，生物制药和其他研究领域中功能性蛋白质产生相关的成本。增加数量的数量将大大减轻此问题。这可以在细菌中通过表达蛋白质作为融合，即感兴趣的蛋白质连接到另一种蛋白质（称为SUMO）来完成。可以从细菌细胞中分离融合，然后可以通过识别SUMO的酶（蛋白酶）去除相扑部分。不幸的是，许多蛋白质需要在哺乳动物细胞中表达，因为细菌没有修饰表达的蛋白质并使其活跃的机械。但是，针对细菌开发的SUMO方法不起作用，但是，对于在真核细胞中表达的蛋白质，因为与细菌不同，这些细胞中含有酶，这些酶可以从融合中从融合中去除SUMO从其他细胞蛋白中纯化。开发了细菌的SUMO系统的救生器提出了设计的特殊突变版本的1）SUMO，不能通过天然酶在细胞内切割，而2）一种突变的蛋白酶，当融合纯化时，可以将突变的Sumo切成细布的蛋白酶，该蛋白酶可以在细胞外切出突变的SUMO。这种组合将允许与细菌系统所提供的相同类型的EMO增强蛋白质产生。