Cytochrome c550 from Thiobacillus versutus: cloning, expression in Escherichia coli, and purification of the heterologous holoprotein.
The gene coding for cytochrome c550 from Thiobacillus versutus, cycA, has been cloned and sequenced. It codes for a protein of 134 amino acids plus a 19-amino-acid-long signal peptide. Both coding and noncoding DNA sequences of the clone are homologous to the Paracoccus denitrificans DNA sequence. An expression vector was constructed by cloning the cycA gene directly behind the lac promoter of pUC. The cycA gene was expressed in Escherichia coli under semianaerobic conditions, and mature holo-cytochrome c550 was isolated with the periplasmic soluble protein fraction. Under both aerobic and anaerobic conditions, significantly less cytochrome c550 was produced. The heterologously expressed cytochrome c550 was isolated and purified to better than 95% purity and was compared with cytochrome c550 isolated and purified from T. versutus. No structural differences could be detected by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis UV-visible light spectroscopy, and 1H nuclear magnetic resonance spectroscopy, indicating that E. coli produces the cytochrome and attaches the heme correctly.
Ubbink
M
M
Gorlaeus Laboratories, Department of Chemistry, Leiden University, The Netherlands.
Van Beeumen
J
J
Canters
G W
GW
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000096:biosynthesis / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006418:Heme / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry; D013855:Thiobacillus / Q000235:genetics*
D001059:Apoproteins; D003574:Cytochrome c Group; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D006418:Heme; C031732:cytochrome C-550