Reconstitution of rabbit skeletal muscle troponin from the recombinant subunits all expressed in and purified from E. coli.
Three subunits of rabbit skeletal muscle troponin were expressed in and purified from Escherichia coli. The procedures were optimized, and the reconstituted troponin complex is highly homogeneous, stable, and obtainable in large quantities, allowing us to conduct crystallization studies of the troponin complex. The three subunits expressed and purified are beta-TnT(N'-208), TnI(C64A, C133S), and the wild type TnC. beta-TnT(N'-208) is a 25 kDa fragment of beta-troponin T, which consists of 208 amino acids and lacks 58 residues in the N-terminal variable region. TnI(C64A, C133S) is a mutant troponin I, in which Cys-64 and Cys-133 are replaced by Ala and Ser, respectively. Each subunit was separately expressed in E. coli, purified by column chromatography including HPLC, and reassembled to form troponin complex. The reconstituted troponin complex was not distinguishable from authentic troponin prepared from rabbit skeletal muscle; the acto-S1 ATPase rate, as well as the superprecipitation, was calcium-sensitive. Small flat crystals up to 0.2 mm long have been reproducibly obtained in preliminary crystallization trials.
Fujita-Becker
S
S
European Molecular Biology Laboratory, Desy, Hamburg, Germany.
Kluwe
L
L
Miegel
A
A
Maeda
K
K
Maéda
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014336:Troponin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014336:Troponin; D009218:Myosins