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 screening of cells that have been transformed with myosin expression constructs and allows the rapid purification of recombinant myosins. Depletion of cellular ATP is used to recruit most of the endogenous and recombinant myosin into a rigor-like complex with actin. Following cell lysis the insoluble actomyosin complex is precipitated by centrifugation, washed, and Mg(2+)-ATP is added to extract the recombinant protein from the pellet. More than 90% of the protein in the resulting supernatant corresponds to actin, myosin, and the recombinant myosin fragments. Therefore, it is easy to detect any differences in expression level between individual myosin constructs on SDS-polyacrylamide gels. Additionally, the dependence of expression on external factors, such as cell density, can be readily determined. Furthermore, the presence of a band corresponding to the recombinant protein indicates that the overexpressed protein has at least some of the functional properties that are characteristic for a myosin motor. This rapid and selective extraction protocol can also be utilized to facilitate the purification of recombinant myosin motors on a preparative scale and has proved particularly useful in the purification of myosin head fragments, that are tagged with histidine residues, by Ni(2+)-chelate affinity chromatography.
Manstein
D J
DJ
National Institute for Medical Research, London, UK.
Hunt
D M
DM
D000185:Actinin / Q000096:biosynthesis / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004023:Dictyostelium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D006801:Humans; D007202:Indicators and Reagents; D015879:Myosin Subfragments / Q000096:biosynthesis* / Q000302:isolation & purification; D009218:Myosins / Q000096:biosynthesis* / Q000302:isolation & purification; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D016324:Sequence Tagged Sites
D007202:Indicators and Reagents; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D000185:Actinin; D006639:Histidine; D000255:Adenosine Triphosphate; D009218:Myosins