OVEREXPRESSION OF MYOSIN MOTOR DOMAINS IN DICTYOSTELIUM - SCREENING OF TRANSFORMANTS AND PURIFICATION OF THE AFFINITY TAGGED PROTEIN

The eukaryotic organism Dictyostelium discoideum has become one of the organisms of choice for the overexpression of recombinant myosins and myosin fragments. Here, we describe a protocol that facilitates the s creening of cells that have been transformed with myosin expression co nstructs and allows the rapid purification of recombinant myosins. Dep letion of cellular ATP is used to recruit most of the endogenous and r ecombinant myosin into a rigor-like complex with actin. Following cell lysis the insoluble actomyosin complex is precipitated by centrifugat ion, washed, and Mg2+-ATP is added to extract the recombinant protein from the pellet, More than 90% of the protein in the resulting superna tant corresponds to actin, myosin, and the recombinant myosin fragment s. Therefore, it is easy to detect any differences in expression level between individual myosin constructs on SDS-polyacrylamide gels. Addi tionally, the dependence of expression on external factors, such as ce ll density, can be readily determined. Furthermore, the presence of a band corresponding to the recombinant protein indicates that the overe xpressed protein has at least some of the functional properties that a re characteristic for a myosin motor. This rapid and selective extract ion protocol can also be utilized to facilitate the purification of re combinant myosin motors on a preparative scale and has proved particul arly useful in the purification of myosin head fragments, that are tag ged with histidine residues, by Ni2+-chelate affinity chromatography.