High yield of Methylophilus methylotrophus cytochrome c by coexpression with cytochrome c maturation gene cluster from Escherichia coli.
Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c" isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS-PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and (1)H-NMR spectroscopy. The success in expressing the mature cytochrome c in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.
Price
N J
NJ
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal.
Brennan
L
L
Faria
T Q
TQ
Vijgenboom
E
E
Canters
G W
GW
Turner
D L
DL
Santos
H
H
D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular / Q000379:methods*; D003574:Cytochrome c Group / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D020630:Methylophilus methylotrophus / Q000235:genetics*; D005810:Multigene Family; D011994:Recombinant Proteins / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
D003574:Cytochrome c Group; D011994:Recombinant Proteins; C076615:cytochrome c''; C017165:cytochrome c(3)