EXPRESSION OF MUTANT VERTEBRATE MYOSIN I'S

肌球蛋白I广泛表达于不同的组织中， 系统发育背景 在肠上皮刷状缘， 肌球蛋白I定位于微绒毛中，在那里它桥接差距 在细胞膜和肌动蛋白束之间。 肌球蛋白I也定位于 靠近迁移细胞活跃的褶皱边缘。 我们使用 杆状病毒/Sf9系统表达全长鸡刷状缘 肌球蛋白I重链（BBMI HC）与钙调蛋白（CaM）一起沿着。 脊椎动物 肌球蛋白I是组成性活性的，而肌球蛋白I是来自低水平的 真核生物如阿米巴和网骨藻需要 位于肌球蛋白头部结构域的丝氨酸磷酸化， 活动 脊椎动物和阿米巴肌球蛋白I的序列比对 揭示了大多数脊椎动物蛋白质都带负电荷， 在所述阿米巴原虫蛋白具有所述氨基酸序列的位置处的氨基酸， 磷酸化丝氨酸 我们正在探索，使用网站导向 诱变和杆状病毒/Sf9系统，是否带负电荷 在这个网站是必不可少的肌动蛋白激活MgATPase的活性， 离体运动 全长鸡BBMI HC已与钙调蛋白共表达， 在杆状病毒/Sf9细胞系统中使用pVL1393作为重组质粒， 转移载体 从感染的细胞中提取可溶性BBMI 使用ATP和高速离心。 表达的BBMI结合至 肌动蛋白以ATP依赖的方式，我们用来帮助 洁净. 一种针对羧基末端14个氨基的抗体 BBMI HC序列的氨基酸用于免疫沉淀BBMI或 捕获它用于体外运动研究。表达的肌球蛋白I 肌动蛋白丝转位的速度与 组织纯化肌球蛋白I。 肌动蛋白丝的运动， 表达的BBMI被Ca ++和原肌球蛋白抑制， 从组织中纯化的BBMI的特征。 我们目前正在 表达羧基末端的FLAG标记的BBMI重链， 进一步研究。

Myosin I's are widely expressed in different tissues and across broad phylogenetic backgrounds. In intestinal epithelial brush borders, myosin I is localized in the microvillus where it bridges the gap between the membrane and the actin bundles. Myosin I is also localized near the actively ruffling edges of migrating cells. We are using the Baculovirus/Sf9 system to express full-length chicken brush border myosin I heavy chain (BBMI HC) along with calmodulin (CaM). Vertebrate myosin I's are constitutively active whereas myosin I's from low eukaryotes such as Acanthamoeba and Dictyostelium require phosphorylation at a serine located in the myosin head domain for activity. Sequence alignments of vertebrate and Acanthamoeba myosin I's reveal that most of the vertebrate proteins have a negatively charged amino acid at the position where the Acanthamoeba protein has the phosphorylatable serine. We are exploring, using site-directed mutagenesis and the Baculovirus/Sf9 system, whether a negative charge at this site is essential for actin-activated MgATPase activity and in vitro motility. Full-length chicken BBMI HC has been coexpressed with calmodulin as light chains in the Baculovirus/Sf9 cell system using pVL1393 as the transfer vector. Soluble BBMI was extracted from the infected cells using ATP and high speed centrifugation. The expressed BBMI binds to actin in an ATP-dependent manner, a property we used to aid in purification. An antibody raised against the carboxyl terminal 14 amino acids of the BBMI HC sequence was used to immunoprecipitate BBMI or capture it for in vitro motility studies. The expressed myosin I translocates actin filaments at a rate indistinguishable from that of tissue purified myosin I. The movement of actin filaments by the expressed BBMI is inhibited by Ca++ and by tropomyosin, which is characteristic of BBMI purified from tissue. We are currently expressing a carboxyl terminal, FLAG-tagged, BBMI heavy chain for further studies.