title: Assembly of functional skeletal muscle troponin complex in Escherichia coli.
abstract: The production of multi-subunit proteins of eukaryotic origin in Escherichia coli usually relies on the different subunits being expressed individually and the protein being reassembled in vitro. Here we describe the construction and characterization of plasmids capable of coexpressing the three subunits of chicken skeletal muscle troponin complex in E. coli. We demonstrate that the troponin subunits assembled in the cytoplasm of E. coli cell are fully functional. The troponin complex was purified to homogeneity in high yields. When reconstituted into actin filaments, the complex assembled in vivo was capable of regulating the myosin ATPase with a calcium dependence that was identical to the complex reconstituted in vitro. These results demonstrate that the coexpression of the subunits of a protein complex can prevent the accumulation of denatured proteins in inclusion granules.
authors.affiliation: Dept. Bioquímica, Universidade de São Paulo, Brazil.
authors.forename: B
authors.initials: B
authors.lastname: Malnic
authors.forename: F C
authors.initials: FC
authors.lastname: Reinach
mesh_terms: D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D009132:Muscles / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010957:Plasmids; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014336:Troponin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019209:Troponin C; D019210:Troponin I
chemical_list: D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D009218:Myosins