This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ubbink, M Articles by Canters, G W Search for Related Content PubMed PubMed Citation Articles by Ubbink, M Articles by Canters, G W

research-article

Cytochrome c550 from Thiobacillus versutus: cloning, expression in Escherichia coli, and purification of the heterologous holoprotein.

Gorlaeus Laboratories, Department of Chemistry, Leiden University, The Netherlands.

ABSTRACT

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.

This article has been cited by other articles:

• Diaz-Moreno, I., Diaz-Quintana, A., De la Rosa, M. A., Ubbink, M. (2005). Structure of the Complex between Plastocyanin and Cytochrome f from the Cyanobacterium Nostoc sp. PCC 7119 as Determined by Paramagnetic NMR: THE BALANCE BETWEEN ELECTROSTATIC AND HYDROPHOBIC INTERACTIONS WITHIN THE TRANSIENT COMPLEX DETERMINES THE RELATIVE ORIENTATION OF THE TWO PROTEINS. J. Biol. Chem. 280: 18908-18915 [Abstract] [Full Text]  
• Hasegawa, J., Shimahara, H., Mizutani, M., Uchiyama, S., Arai, H., Ishii, M., Kobayashi, Y., Ferguson, S. J., Sambongi, Y., Igarashi, Y. (1999). Stabilization of Pseudomonas aeruginosa Cytochrome c551 by Systematic Amino Acid Substitutions Based on the Structure of Thermophilic Hydrogenobacter thermophilus Cytochrome c552. J. Biol. Chem. 274: 37533-37537 [Abstract] [Full Text]  
• Baker, S. C., Ferguson, S. J., Ludwig, B., Page, M. D., Richter, O.-M. H., van Spanning, R. J. M. (1998). Molecular Genetics of the Genus Paracoccus: Metabolically Versatile Bacteria with Bioenergetic Flexibility. Microbiol. Mol. Biol. Rev. 62: 1046-1078 [Abstract] [Full Text]  
• Keightley, J. A., Sanders, D., Todaro, T. R., Pastuszyn, A., Fee, J. A. (1998). Cloning and Expression in Escherichia coli of the Cytochrome c552 Gene from Thermus thermophilus HB8. EVIDENCE FOR GENETIC LINKAGE TO AN ATP-BINDING CASSETTE PROTEIN AND INITIAL CHARACTERIZATION OF THE cycA GENE PRODUCTS. J. Biol. Chem. 273: 12006-12016 [Abstract] [Full Text]