Recombinant expression of Mus musculus myoglobin.
The cDNA encoding for Mus musculus myoglobin (Mb) was amplified using standard RT-PCR techniques and cloned in an appropriate bacterial expression vector. For the first time, mouse Mb was recombinantly expressed in Escherichia coli cells, BL21(DE3), and purified in sufficient amounts to carry out a preliminary characterization. As shown by mass spectrometry, the protein is found in complex with glutathione, which binds the Cys residue in the topological position E9, in the proximity of the heme group. In recombinant murine Mb, azide affinities are only slightly dependent on the Cys(E9) oxidation state. This suggests that Cys(E9) does not provide a relevant contribution for the stabilization of ligands bound to the heme iron atom. Recombinant expression of M. musculus Mb might have an important role in order to investigate the eventual involvement of Cys(E9) in the new physiological roles proposed for Mb.
Bianchi
Marzia
M
CNR, Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Catholic University of the Sacred Heart, Largo Francesco Vito 1, 00168 Rome, Italy.
Clementi
Maria Elisabetta
ME
Maras
Bruno
B
Schininà
Maria Eugenia
ME
Bozzi
Manuela
M
Giardina
Bruno
B
Brancaccio
Andrea
A
D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology; D009211:Myoglobin / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D001386:Azides; D009211:Myoglobin; D011994:Recombinant Proteins; D005978:Glutathione; D010100:Oxygen