Expression Studies Of Other Unconventional Myosins

我们正在使用杆状病毒/Sf9系统表达其他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，在初步实验中，我们无法检测到任何镁ATPase活性或肌动蛋白结合。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：一个包含通过轻链结合基序延伸的氨基末端的片段(长S1)，一个缺失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。肌球蛋白IX是一种不常见的肌球蛋白，其尾部含有一个GTP酶激活区。肌球蛋白是单头的，特征不是很好。我们已经开始表达这种肌球蛋白的头部片段，但发现该蛋白似乎没有很好地表达。

We are using the baculoviral/Sf9 systems to express several other 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 or actin binding in preliminary experiments. 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 attemping 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 IX is an unusual myosin which contains a GTPase activating domain in its tail. The myosin is single-headed and is not well characterized. We have begun the expression of head fragments of this myosin but find that the protein does not appear to express well.