EXPRESSION OF MUTANT VERTEBRATE MYOSIN I'S

突变脊椎动物肌球蛋白 I 的表达

• 批准号: 5203560
• 负责人: F WANG
• 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/5203560
• 关键词: Baculoviridae; Sf9 cell line; actins; adenosine triphosphate; calcium; calmodulin; centrifugation; enzyme activity; gene expression; gene mutation; immunoprecipitation; mutant; myosins; phosphoproteins; protein sequence; serine; site directed mutagenesis; transfection /expression vector; tropomyosin

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.

肌球蛋白I在不同的组织和广泛的组织中广泛表达 系统发育背景。在肠道上皮刷状缘， 肌球蛋白I定位于连接缝隙的微绒毛中 在膜和肌动蛋白束之间。肌球蛋白I也是局部化的 靠近迁移细胞活跃的褶皱边缘。我们使用的是 杆状病毒/Sf9系统表达全长鸡刷边界 肌球蛋白I重链(BBMI、HC)和钙调蛋白(CaM)。脊椎动物 肌球蛋白I是结构性活性的，而肌球蛋白I是从低 真核生物，如棘阿米巴和网柄金龟子，需要 位于肌球蛋白头部结构域的丝氨酸的磷酸化 活动。脊椎动物与棘阿米巴肌球蛋白I‘s的序列比对 揭示了大多数脊椎动物的蛋白质都带有负电荷 棘阿米巴蛋白具有 可磷酸化丝氨酸。我们正在探索，使用站点定向 突变与杆状病毒/Sf9系统，是否带负电荷 对于肌动蛋白激活的镁ATPase活性和在 体外运动性。 全长鸡BBMI HC与钙调蛋白共表达为 以pVL1393为靶标的杆状病毒/Sf9细胞系统中的轻链 传递向量。从感染细胞中提取可溶性BBMI。 采用三磷酸腺苷和高速离心法。表达的BBMI结合于 肌动蛋白以一种依赖于ATP的方式，这是我们过去帮助 净化。针对羧基末端14氨基的抗体 BBMI HC序列的酸用于免疫沉淀BBMI或 捕获它以进行体外运动性研究。表达的肌球蛋白I 移位肌动蛋白细丝的速度与 组织纯化的肌球蛋白I.肌动蛋白细丝的运动 表达的BBMI被钙离子和原肌球蛋白抑制，这是 从组织中提纯的BBMI的特性。我们目前正在 表达标志标记的羧基末端的BBMI重链 进一步的研究。