ArticleJournal of muscle research and cell motility1995
Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein.
Article in Journal of muscle research and cell motility, 1995. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 27 papers.
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Who cites it
27 citing papers in PubMed, 77 citations in OpenAlex.
- The filopodial myosin DdMyo7 is a slow, calcium-regulated motor.The Journal of biological chemistry · 2025Article
- Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke.International journal of molecular sciences · 2020Article
- The Conserved Lysine-265 Allosterically Modulates Nucleotide- and Actin-binding Site Coupling in Myosin-2.Scientific reports · 2017Article
- Article
- Metal switch-controlled myosin II from Dictyostelium discoideum supports closure of nucleotide pocket during ATP binding coupled to detachment from actin filaments.The Journal of biological chemistry · 2013Article
- Allosteric communication in Dictyostelium myosin II.Journal of muscle research and cell motility · 2012Article
- Structural basis for the allosteric interference of myosin function by reactive thiol region mutations G680A and G680V.The Journal of biological chemistry · 2011Article
- Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.The Journal of biological chemistry · 2011Article
- Myosin cleft closure determines the energetics of the actomyosin interaction.FASEB journal : official publication of the Federation of American Societies for Experimental Biology · 2011Article
- Unconventional secretion of AcbA in Dictyostelium discoideum through a vesicular intermediate.Eukaryotic cell · 2010Article
- Insights into the importance of hydrogen bonding in the gamma-phosphate binding pocket of myosin: structural and functional studies of serine 236.Biochemistry · 2010Article
- A myosin IK-Abp1-PakB circuit acts as a switch to regulate phagocytosis efficiency.Molecular biology of the cell · 2010Article
- The mechanism of pentabromopseudilin inhibition of myosin motor activity.Nature structural & molecular biology · 2009Article
- Enzyme kinetics above denaturation temperature: a temperature-jump/stopped-flow apparatus.Biophysical journal · 2006Article
- Genetic evidence that the acyl coenzyme A binding protein AcbA and the serine protease/ABC transporter TagA function together in Dictyostelium discoideum cell differentiation.Eukaryotic cell · 2006Article
- Crystal structure of the GTPase domain of rat dynamin 1.Proceedings of the National Academy of Sciences of the United States of America · 2005Article
- Conformational changes in actin-myosin isoforms probed by Ni(II).Gly-Gly-His reactivity.Journal of muscle research and cell motility · 2004Article
- Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium.EMBO reports · 2002Article
- Crystal structure of the motor domain of a class-I myosin.The EMBO journal · 2002Article
- Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.The EMBO journal · 2001Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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