Heterologous Expression in Pseudomonas aeruginosa and Purification of the 9.2-kDa c-Type Cytochrome Subunit of p-Cresol Methylhydroxylase

Cronin, Ciarán N.; McIntire, William S.

doi:10.1006/prep.2000.1218

Cited by 12 publications

(10 citation statements)

References 31 publications

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“…The fact that the Pseudomonas putida cytochrome c subunit of p-cresol methylhydroxylase, another flavocytochrome c, was successfully overproduced in Pseudomonas aeruginosa (Cronin and McIntire 2000) after several attempts to produce this subunit in E. coli supported our idea to study overexpression of FCSD genes in one of the photosynthetic bacteria. Likewise, due to the difficult expression of cytochromes c in E. coli, other hosts have been used for expression of cytochromes c genes.…”

Section: Discussionmentioning

confidence: 96%

A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter

Smet

Kostanjevečki

Guisez

et al. 2001

Archives of Microbiology

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Flavocytochrome c-sulfide dehydrogenase (FCSD), an enzyme that catalyzes the reversible conversion of sulfide to elemental sulfur in vitro, is common to bacteria that utilize reduced sulfur compounds as electron donors in the process of carbon dioxide fixation. FCSD is a heterodimer containing two different cofactors, a flavin (FAD) and one or two heme c groups, located on the separate protein subunits. Efforts to produce the holoproteins of the soluble Allochromatium vinosum FCSD and the membrane-bound Ectothiorhodospira vacuolata protein in Escherichia coli using several expression systems were unsuccessful. Although all systems used were able to export the recombinant FCSDs to the periplasm, the proteins did not incorporate heme. In order to develop a new expression system involving photosynthetic hosts (Rhodobacter capsulatus, Rhodobacter sphaeroides and Ect. vacuolata), plasmid mobilisation from E. coli donors was studied. In the search for efficient promoters for such hosts, a system was developed combining the broad-host-range plasmid pGV910 and the promoter of the A. vinosum RuBisCo gene, rbcA. Conjugation was used to enable transfer from the expression plasmid of E. coli into Rba. capsulatus, Rba. sphaeroides strains and into Ect. vacuolata. Both Rhodobacter hosts were able to transcribe the genes coding for FCSD from the rbcA promoter and to produce detectable amounts of recombinant FCSD holoprotein. Western blots showed that the best production was obtained from cells grown photosynthetically on malate or acetate with sulfide. This system may prove to be of general use for the production of recombinant c-type cytochromes in homologous or related host systems.

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Section: Discussionmentioning

confidence: 96%

A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter

Smet

Kostanjevečki

Guisez

et al. 2001

Archives of Microbiology

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“…The PCR product was purified using a spin‐column kit (Qiagen, Inc., Valencia, CA, USA). The PCR product and the expression vector pUCPNde [17] were digested with Nde I and Xma I as directed by the manufacturer (Promega, Inc., Madison, WI, USA). The digested PCR product and expression vector were ligated and transformed into frozen competant Escherichia coli strain DH5αF′IQ cells (Life Technologies) using standard methods [18].…”

Section: Dna Techniquesmentioning

confidence: 99%

“…The resulting plasmids, pUCYPKR, pUCYPKA, and pUCYPKY, were transformed into cells [20]. The plasmids pUCYP2, pUCYPKR, pUCYPKA, and pUCYPKY were each introduced into Pseudomonas aeruginosa strain PAO‐ Lac I by electroporation using an Electroporator 2510 (Eppendorf Co., Westbury, NY, USA) as described by Cronin and McIntire [17].…”

Section: Dna Techniquesmentioning

confidence: 99%

“…Cells of P. aeruginosa PAO lac I‐containing plasmids pUCYP2, pUCYPKR, pUCYPKA, or pUCYPKY were grown and periplasmic extracts prepared as described by Cronin and McIntire [17]. The periplasmic extract was dialyzed in Union Carbide 5 cm‐wide dialysis bags ( m cutoff < 25 kDa) overnight at 5 °C against 6 L of KP i buffer (50 m m potassium phosphate, pH 7.5).…”

Section: Growth Of Cells and Purification Of Cytochrome P460mentioning

confidence: 99%

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Cytochrome P460 of Nitrosomonas europaea

Bergmann

Hooper

2003

European Journal of Biochemistry

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The heme of cytochrome P460 of Nitrosomonas europaea, which is covalently crosslinked to two cysteines of the polypeptide as with all c‐type cytochromes, has an additional novel covalent crosslink to lysine 70 of the polypeptide [Arciero, D.M. & Hooper, A.B. (1997) FEBS Lett.410, 457–460]. The protein can catalyze the oxidation of hydroxylamine. The gene for this protein, cyp, was expressed in Pseudomonas aeruginosa strain PAO lacI, resulting in formation of a holo‐cytochrome P460 which closely resembled native cytochrome P460 purified from N. europaea in its UV‐visible spectroscopic, ligand binding and catalytic properties. Mutant versions of cytochrome P460 of N. europaea in which Lys70 70 was replaced by Arg, Ala, or Tyr, retained ligand‐binding ability but lost catalytic ability and differed in optical spectra which, instead, closely resembled those of cytochromes c′. Tryptic fragments containing the c‐heme joined only by two thioether linkages were observed by MALDI‐TOF for the mutant cytochromes P460 K70R and K70A but not in wild‐type cytochrome P460, consistent with the structural modification of the c‐heme only in the wild‐type cytochrome. The present observations support the hypothesized evolutionary relationship between cytochromes P460 and cytochromes c′ in N. europaea and M. capsulatus[Bergmann, D.J., Zahn, J.A., & DiSpirito, A.A. (2000) Arch. Microbiol. 173, 29–34], confirm the importance of a heme‐crosslink to the spectroscopic properties and catalysis and suggest that the crosslink might form auto‐catalytically.

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“…Other hosts have been used for heterologous expression. [20][21][22] Generally speaking, the covalent attachment of heme to c-type cytochromes must be performed by a dedicated post-translational modification apparatus; these vary in different organisms and subcellular compartments. 7 There are four known post-translational modification protein systems for producing c-type cytochromes.…”

Section: Production Of Cytochromes C Using Cellular Post-translationa...mentioning

confidence: 99%

Cytochrome c as an experimental model protein

Stevens

2011

Metallomics

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Mitochondrial cytochrome c is among the most intensively studied of all proteins. Initial interest was in its role in the respiratory chain and as a model for studies of protein structure, folding and electron transfer. The function of cytochrome c in signalling apoptosis has brought a new wave of research into the protein. Bacterial cytochromes c are more complex and varied in function. This review highlights some of these roles and expands on systems for producing holocytochrome c proteins.

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Heterologous Expression in Pseudomonas aeruginosa and Purification of the 9.2-kDa c-Type Cytochrome Subunit of p-Cresol Methylhydroxylase

Cited by 12 publications

References 31 publications

A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter

A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter

Cytochrome P460 of Nitrosomonas europaea

Cytochrome c as an experimental model protein

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