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.
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