Interactions of SARS-CoV-2 N-protein

SARS-CoV-2 N 蛋白的相互作用

• 批准号: 10263005
• 负责人: PETER SCHUCK
• 金额: $ 23.93万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10263005
• 关键词: 2019-nCoV; Affinity; Antigens; Binding; Biological Assay; Biophysics; COVID-19; Cells; Collaborations; Complex; Fibrosis; Genome; Investigation; Label; Laboratories; Measures; Nucleic Acids; Nucleocapsid; Oligonucleotides; Pathway interactions; Pharmaceutical Preparations; Play; Property; Proteins; RNA Binding; RNA Processing; Reaction; Role; SARS-CoV N protein; Serological; Site; Source; Techniques; Therapeutic; Tissues; Viral; Virus Assembly; Virus Diseases; analytical ultracentrifugation; biophysical analysis; dimer; experimental study; immunoregulation; inhibitor/antagonist; particle; protein expression; sedimentation velocity; small molecule therapeutics; therapeutic target

To allow an immediate start of our investigations we have examined N-protein from several commercial sources and identified suitable protein constructs. Simultaneously we have started to develop in-house protein expression, jointly with the laboratory of Dr. Eugene Valkov. We are currently optimizing solution conditions, and are exploring and implementing purification strategies. We have carried out initial sedimentation velocity analytical ultracentrifugation experiments to assess the oligomeric state and found it to be dimeric, similar to SARS-CoV N-protein. We explored different fluorescent labeling strategies to better measure the affinity of self-association. We have also carried out initial binding experiments with small oligonucleotides, which will help us to optimize the biophysical assays for assembly reactions. In collaboration with Dr. Greg Piszczek we have begun to characterize the thermal properties of the protein and its complexes. We are evaluating the first results.

为了能够立即开始我们的研究，我们检查了几个商业来源的N-蛋白质，并确定了合适的蛋白质结构。与此同时，我们已经开始与尤金·瓦尔科夫博士的实验室合作，开发内部蛋白质表达。我们目前正在优化溶液条件，并正在探索和实施纯化策略。 我们进行了初步的沉降速度分析超速离心实验，以评估寡聚状态，发现它是二聚体，类似于SARS-CoV N蛋白。我们探索了不同的荧光标记策略，以更好地测量自关联的亲和力。我们还进行了与小分子寡核苷酸的初步结合实验，这将有助于我们优化组装反应的生物物理分析。在与Greg Piszczek博士的合作下，我们已经开始表征蛋白质及其复合体的热性质。我们正在评估第一批结果。