Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein

Abstract

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.

Crystal structure of the GTPase domain of rat dynamin 1

“…The structure of the myosin part has been published elsewhere (16). The Nterminally His-tagged 120-kDa fusion protein was overproduced in Dictyostelium AX3-ORF ϩ and purified essentially as described earlier for the myosin part alone (17). Cells were lysed with 0.5% (vol͞vol) Triton X-100 in 20 cell volumes of buffer containing 50 mM Tris (pH 8.0), 2 mM EDTA, 0.2 mM EGTA, 2 mM dithiothreitol (DTT), 5 mM benzamidine, and protease inhibitors.…”

Myosin cleft movement and its coupling to actomyosin dissociation

“…The N-terminal sequence of Md was modified to MDGTP where P corresponds to P3 of the native sequence. The Cterminal sequence was the same as in the M761 construct and contained a His 8 fusion for ease of purification 26,27 . After cloning, the PCR amplified fragment was sequenced.…”

Stabilization of the Actomyosin Complex by Negative Charges on Myosin

“…Cells were harvested at a density of about 5.5 × 10 6 mL -1 by centrifugation for 7 min at 2400 rpm in a Beckman J-6 centrifuge and washed once in phosphate-buffered saline. The histidine-tagged recombinant proteins were purified to homogeneity by binding to actin and release with ATP followed by Ni 2+ -chelate affinity chromatography as described by Manstein and Hunt (18). An average yield of 3 mg of myosin head fragment was obtained per gram of cells.…”