Expression studies of other unconventional myosins

其他非常规肌球蛋白的表达研究

• 批准号: 6967009
• 负责人: JAMES R. SELLERS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/6967009
• 关键词: Baculoviridae; Sf9 cell line; circadian rhythms; deafness; enzyme activity; gene expression; guanine nucleotide binding protein; human genetic material tag; isozymes; labyrinth; myosins; phosphoproteins; phosphorylation; protein isoforms; protein structure function; recombinant proteins; visual photoreceptor

We are using the baculoviral/Sf9 systems to express several unconventional myosins, including myosin III from Limulus, the horseshoe crab, and myosin XV from human. Myosin III is unusual in that there is a kinase domain at the amino-terminus. The myosin is found exclusively in the photoreceptor cells of the visual system. A full-length clone expresses well in Sf9 cells and has been purified to homogeneity. Interestingly, we are unable to detect any MgATPase activity. Recent studies show that it binds only very weakly to actin in either the presence or absence of ATP. The protein is phosphorylated in situ in a circadian manner. We are investigating whether this phosphorylation plays any effect on its activity. Myosin XV is a relatively newly discovered myosin that was identified in mice and humans as a deafness gene. It has a very long proline-rich amino-terminal extension of unknown function which can be alternatively spliced. We are attempting to express three fragments: a fragment that contains the amino-terminus extension through the light chain binding motifs (long S1), a fragment that deletes the alternatively spliced amino-terminus (short S1) and a full-length construct. So far, only the short S1 appears to be expressed in a soluble form. We are currently assessing its enzymatic activity. Myosin VIIb from Drosophila was cloned and we are expressing a fragment containing the head, neck and predicted coiled-coil forming sequences in baculovirus. Preliminary data suggest that the expressed protein is monomeric. It has a Vmax of 6 per sec and a Km of 20 micromolar. We have also expressed a fragment of myosin XVIIIa and are in the process of determining its functional properties. This myosin contains several interesting domains, including an N-terminal KE domain and a PDZ domain. It is alternatively spliced at several places. We are beginning to study the function of the various domains using a combination of biochemical and cell biological techniques

我们正在使用杆状病毒/Sf 9系统表达几种非传统的肌球蛋白，包括来自鲎的肌球蛋白III和来自人类的肌球蛋白XV。肌球蛋白III是不寻常的，因为有一个激酶域的氨基末端。肌球蛋白只存在于视觉系统的感光细胞中。全长克隆在Sf 9细胞中表达良好，并已纯化至均一。有趣的是，我们无法检测到任何MgATPase活性。最近的研究表明，无论ATP存在与否，它都只能很弱地与肌动蛋白结合。蛋白质以昼夜节律的方式原位磷酸化。我们正在研究这种磷酸化是否对其活性有任何影响。肌球蛋白XV是一种相对较新发现的肌球蛋白，在小鼠和人类中被鉴定为耳聋基因。它有一个很长的富含脯氨酸的氨基末端延伸，功能未知，可以选择性剪接。我们正试图表达三个片段：一个片段，含有氨基末端延伸通过轻链结合基序（长S1），一个片段，删除可变剪接的氨基末端（短S1）和全长构建体。到目前为止，只有短的S1似乎以可溶形式表达。我们目前正在评估其酶活性。从果蝇的肌球蛋白VIIb克隆，我们表达的片段包含头部，颈部和预测的卷曲螺旋形成序列的杆状病毒。初步数据表明，表达的蛋白质是单体。它的Vmax为每秒6，Km为20微摩尔。我们还表达了肌球蛋白XVIIIa的片段，并正在确定其功能特性。该肌球蛋白含有几个有趣的结构域，包括N-末端KE结构域和PDZ结构域。它在几个地方交替剪接。我们正开始使用生物化学和细胞生物学技术相结合的方法来研究各个结构域的功能