Expression studies of other unconventional myosins

我们正在使用杆状病毒/Sf9系统表达几种非常规肌球蛋白，包括河蟹的肌球蛋白III，马蹄蟹的肌球蛋白III，以及人类的肌球蛋白XV。肌球蛋白III的不同寻常之处在于，在氨基末端有一个激活域。肌球蛋白只存在于视觉系统的感光细胞中。一个全长克隆在Sf9细胞中表达良好，并已纯化为均一。有趣的是，我们无法检测到任何镁ATPase活性。最近的研究表明，在有或没有ATP的情况下，它与肌动蛋白的结合都非常弱。该蛋白质以昼夜节律的方式被原位磷酸化。我们正在调查这种磷酸化是否会对其活性产生任何影响。肌球蛋白XV是一种相对较新发现的肌球蛋白，在老鼠和人类中被鉴定为耳聋基因。它有一个很长的富含Pro的未知功能的氨基末端延伸，可以选择性地剪接。我们试图表达三个片段：一个通过轻链结合基序包含氨基末端延伸的片段(长S1)，一个删除可选择性剪接的氨基末端的片段(短S1)和一个全长结构。到目前为止，只有短的S1似乎以可溶的形式表达。我们目前正在评估其酶活性。从果蝇中克隆了肌球蛋白VIIB，并在杆状病毒中表达了包含头部、颈部和预测的卷曲形成序列的片段。初步数据表明，表达的蛋白质是单体。它的Vmax为每秒6，公里为20微摩尔。我们还表达了一段肌球蛋白XVIIIa，并正在确定其功能性质。该肌球蛋白含有几个有趣的结构域，包括N-末端的KE结构域和PDZ结构域。它在几个地方交替拼接。我们开始结合生物化学和细胞生物学技术来研究不同结构域的功能。

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