CORE--MOTOR PROTEIN PRODUCTION

核心B的功能是为各种项目提供蛋白质和定制合成有机化学品。在过去的四年里，核心已经生产和分配了许多蛋白质。它们是由两名技术人员制作的，他们是最近去读研究生的亚伦·鲁比和玛丽亚·马图斯卡。生产了激动素和NCD单体，并提供给库克、门德尔森·弗莱特里克和海史密斯。NCD二聚体被生产并分发给Fletterick、Cooke和Mendelson。肌动蛋白和肌球蛋白亚片段分布于Spudich和Highsmith。Aaron Ruby参与了一系列激动素突变体的生产，这些突变体在淡水河谷实验室中使用，也将用于其他项目。微管被生产并分发给库克、海史密斯、淡水河谷和门德尔森。这些蛋白质的集中生产大大加快了该计划取得的进展。每个实验室生产这些蛋白质将是一个耗时的过程，这将浪费时间和物资。CORE还生产了一种GTP的荧光模拟物，用于淡水河谷和库克实验室。要生产的蛋白质分为两类，一类是从动物组织中提取的天然蛋白质，另一类是在大肠杆菌、盘状网柄菌SF-9或毕赤酵母系统中克隆和表达的蛋白质。第一类包括肌动蛋白、肌动蛋白、微管和肌球蛋白。这些蛋白质的纯化遵循既定的程序，通常这些蛋白质将被两个或更多的项目使用。在可行的情况下，蛋白质将被克隆并在大肠杆菌中表达。通常，由于良好的生长和表达特性，野生型和突变蛋白都将在项目中使用。野生型和突变型肌球蛋白亚段的表达和纯化都将来自网柄菌属，该蛋白的克隆将在Spudich计划中构建。一旦一种给定的蛋白质在Dictyostelials中的表达被确定，这种Dictyostelius菌株将由在Spudich实验室操作的Core的人员生产。其他蛋白质的结构将来自淡水河谷或弗莱特里克实验室。

The function of Core B is to provide proteins and custom synthesized organic chemicals t the various projects. During the past four years the core has produced and distributed a number of proteins. These were produced by two technicians, Aaron Ruby, who recently left for graduate school, and Marija Matuska. Both kinesin and NCD monomers were produced and provided to Cooke, Mendelson Fletterick and Highsmith. NCD dimers were produced and distributed to Fletterick, Cooke, and Mendelson. Actin and myosin subfragments were distributed to Spudich and Highsmith. Aaron Ruby participated in the production of a series of kinesin mutants that were used in the Vale laboratory and will also be available for the other projects. Microtubules were produced and distributed to Cooke, Highsmith, Vale and Mendelson. The central production of these proteins has greatly accelerated the progress made by the program. The production of these proteins by each laboratory would be a time consuming process, that would waste time and supplies. The core also produced a fluorescent analog of GTP for use in the Vale and Cooke laboratories. The proteins to be produced fall into two categories, native proteins made from animal tissues, and proteins cloned and expressed in E. coli., Dictyostelium discoidium SF-9 or pichia systems. The first group includes kinesins, actin and microtubules and myosin. These protein purification follow established procedures, and usually the proteins will be used by two or more of the projects. Where feasible, proteins will be cloned and expressed in E. coli. Typically, due to favorable growth and expression characteristics, both wild type and mutant proteins will be used in the projects. Expression and purification of both wild type and mutant myosin subfragments will be from dictyostelium, and clones for this proteins will be constructed in the Spudich project. Once the expression of a given protein in the dictyostelium has been established, this strain of dictyostelium will be produced by personnel from the Core operating in the Spudich lab. Constructs for other proteins will derived from the Vale or Fletterick labs.