Heterologous expression of azurin from Pseudomonas aeruginosa in the yeast Pichia pastoris.

Azurin, which is a bacterial secondary metabolite has been attracted as a potential anticancer agent in recent years because induced death of cancer cells and inhibited their growth. In this study, the production of azurin under the control of the alcohol oxidase promoter which is frequently used in the Pichia pastoris expression system was performed. The azurin gene amplified from Pseudomonas aeruginosa genomic DNA and inserted into the pPICZαA was cloned in Escherichia coli cells. Then, a linearized recombinant vector was transferred to the P. pastoris X-33 cells. Antibiotic resistance test and colony PCR were performed for the selection of multicopy transformants. Protein expression capacities of selected transformants were compared at the end of 48 h incubation. Both extracellular and intracellular protein expressions were observed in all of them by Western blot analysis. The relative expression levels of both intracellular and extracellular protein that belongs to the first clone were higher than the others. On the other hand, it was seen that the 4th clone had the highest protein secretion ability. The molecular mass of the extracellular azurin protein which is produced by recombinant clones was found to be about 20 kDa. This is the first report on azurin expression in P. pastoris.

Escherichia coli Nissle 1917 targets and restrains mouse B16 melanoma and 4T1 breast tumors through expression of azurin protein.

Many studies have demonstrated that intravenously administered bacteria can target and proliferate in solid tumors and then quickly be released from other organs. Here, we employed the tumor-targeting property of Escherichia coli Nissle 1917 to inhibit mouse B16 melanoma and 4T1 breast tumors through the expression of azurin protein. For this purpose, recombinant azurin-expressing E. coli Nissle 1917 was developed. The levels of in vitro and in vivo azurin secretion in the engineered bacterium were determined by immunochemistry. Our results demonstrated that B16 melanoma and orthotopic 4T1 breast tumor growth were remarkably restrained and pulmonary metastasis was prevented in immunocompetent mice. It is worth noting that this therapeutic effect partially resulted from the antitumor activity of neutrophils and lymphocytes due to inflammatory responses caused by bacterial infections. No toxicity was observed in the animal during the experiments. This study indicates that E. coli Nissle 1917 could be a potential carrier to deliver antitumor drugs effectively for cancer therapy.

Overexpression of rusticyanin in Acidithiobacillus ferrooxidans ATCC19859 increased Fe(II) oxidation activity.

A wide-host-range plasmid pTRUS containing a homologous rus gene under the control of Ptac promoter was constructed and transferred into Acidithiobacillus ferrooxidans ATCC19859 using conjugation gene transfer method to generate the engineered strain of A. ferrooxidans(pTRUS). The plasmid-based recombinant rus gene was successfully expressed. According to the results of real-time quantitative PCR assay, not only the transcription level of recombinant rus gene but also the transcription levels of the other genes of rus operon (cyc1, orf, coxB, coxA) were increased in A. ferrooxidans(pTRUS). The ferrous ion (Fe(2+)) oxidation activity was increased in A. ferrooxidans(pTRUS).

Cu(A) centers and their biosynthetic models in azurin.

Cu(A) is a binuclear copper center that functions as an electron transfer agent, cycling between a reduced Cu(I)Cu(I) state and an oxidized mixed-valence Cu(+1.5)...Cu(+1.5) state. The copper ions are bridged by two cysteine thiolate ligands and form a copper-copper bond, the first reported of its kind in Nature. Such a "diamond-core" Cu(2)S(Cys)(2) structure allows an unpaired electron to be completely delocalized over the two copper ions and contributes to its highly efficient electron transfer properties. This review provides accounts of how the Cu(A) center was structurally characterized and highlights its salient spectroscopic properties. In the process, it introduces the Cu(A) center in four different systems-native protein systems, soluble protein truncates of native proteins, synthetic models using organic molecules, and biosynthetic models using proteins as ligands-with a greater emphasis on biosynthetic models of Cu(A), especially on new, deeper insights gained from their studies.

Reorganization energy of the CuA center in purple azurin: impact of the mixed valence-to-trapped valence state transition.

Mixed valence (MV) coordination compounds play important roles in redox reactions in chemistry and biology. Details of the contribution of a mixed valence state to protein electron transfer (ET) reactivity such as reorganization energy, however, have not been experimentally defined. Herein we report measurements of reorganization energies of a binuclear CuA center engineered into Pseudomonas aeruginosa azurin that exhibits a reversible transition between a totally delocalized MV state at pH 8.0 and a trapped valence (TV) state at pH 4.0. The reorganization energy of a His120Ala variant of CuA azurin that displays a TV state at both the above pH values has also been determined. We found that the MV-to-TV state transition increases the reorganization energy by 0.18 eV, providing evidence that the MV state of the CuA center has lower reorganization energy than its TV counterpart. We have also shown that lowering the pH from 8.0 to 4.0 results in a similar (approximately 0.4 eV) decrease in reorganization energy for both blue (type 1) and purple (CuA) azurins, even though the reorganization energies of the two different copper centers are different at a given pH. These results suggest that the MV state plays only a secondary role in modulation of the ET reactivity via the reorganization energy, as compared to that of the driving force.

Heterologous metalloprotein biosynthesis in Escherichia coli: conditions for the overproduction of functional copper-containing nitrite reductase and azurin from Alcaligenes xylosoxidans.

This paper reports on the optimization of conditions for the overproduction and isolation of two recombinant copper metalloproteins, originally encoded on the chromosome of the dentrifying soil bacterium Alcaligenes xylosoxidans, in the heterologous host Escherichia coli. The trimeric enzyme nitrite reductase (NiR) contains both type-1 and type-2 Cu centres, whilst its putative redox partner, azurin I, is monomeric and has only a type-1 Cu centre. Both proteins were processed and exported to the periplasm of E. coli, which is consistent with their periplasmic location in their native host A. xylosoxidans. NiR could be readily purified from the periplasmic fraction of E. coli but the enzyme as isolated possessed only type-1 Cu centres. The type-2 Cu centre could be fully reconstituted by incubation of the periplasmic fraction with copper sulfate prior to enzyme purification. Azurin I could only be isolated with a fully occupied type-1 centre when isolated from the crude cell extract but not after isolation from the periplasmic fraction, suggesting loss of the copper due to proteolysis. Based on a number of criteria, including spectroscopic, mass spectrometric, biochemical and structural analyses, both recombinant proteins were found to be indistinguishable from their native counterparts isolated from A. xylosoxidans. The findings of this work have important implications for the overproduction of recombinant metalloproteins in heterologous hosts.

Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase.

The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic-strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.

Localization of periplasmic redox proteins of Alcaligenes faecalis by a modified general method for fractionating gram-negative bacteria.

A lysozyme-osmotic shock method is described for fractionation of Alcaligenes faecalis which uses glucose to adjust osmotic strength and multiple osmotic shocks. During phenylethylamine-dependent growth, aromatic amine dehydrogenase, azurin, and a single cytochrome c were localized in the periplasm. Their induction patterns are different from those for the related quinoprotein methylamine dehydrogenase and its associated redox proteins.

Sequence and expression of the rusticyanin structural gene from Thiobacillus ferrooxidans ATCC33020 strain.

The periplasmic blue copper protein rusticyanin is thought to play an important role in iron oxidation by Thiobacillus ferrooxidans. We present the sequence of the gene, rus, encoding rusticyanin together with about 1.4 kb of upstream and 0.3 kb of downstream DNA. The rus gene is unique to T. ferrooxidans. Evidence is presented that it is the last gene of an operon and that it can be transcribed from its own promoter. In ATCC33020 strain, rusticyanin is synthesized in ferrous iron but also in sulfur growth conditions suggesting that it could play a role in both energetic metabolisms. The rus gene transcribed from a vector promoter in Escherichia coli leads to the production of a processed aporusticyanin in the periplasmic space, indicating that its signal sequence is correctly recognized by the secretion machinery and the signal peptidase of E. coli.

Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli.

The azurin-encoding azu gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. A purification procedure was developed to isolate the azurin obtained from the E. coli cells. No differences were observed between azurins isolated from P. aeruginosa and E. coli. A non-reconstitutable azurin-like protein, azurin*, with a spectral ratio (A625/A280) less than 0.01 could be separated from holo-azurin with a spectral ratio of 0.58 (+/- 0.01). The properties of azurin* were examined by electrophoretic (SDS-PAGE and IEF) and spectroscopic (UV/vis, 1H-NMR, static and dynamic fluorescence) techniques, and compared to the properties of holo-azurin and apo-azurin. Azurin* resembles apo-azurin (same pKa* values of His-35 and His-117, same fluorescence characteristics). However, it has lost the ability to bind Cu-ions. It is tentatively concluded that azurin* is a chemically modified form of azurin, the modification possibly being due to oxidation of the ligand residue Cys-112 or the formation of a chemical bond between the ligand residues Cys-112 and His-117. In agreement with previous results from Hutnik and Szabo (Biochemistry (1989) 28, 3923-3934), fluorescence experiments show that the heterogeneous fluorescence decay observed for holo-azurin is not due to the presence of azurin*, but most likely originates from conformational heterogeneity of the holo-azurin.

The azurin gene from Pseudomonas aeruginosa. Cloning and characterization.

We have cloned and sequenced the Pseudomonas aeruginosa azurin structural gene and its flanking regions. The DNA sequence predicts a pre-protein with a signal peptide of 19 amino acids followed by the 128-amino-acid mature azurin protein. Nuclease-S1 mapping and primer elongation experiments indicated two 5' termini of the azurin transcript. The major transcript of the azurin gene is initiated around 35 base pairs upstream from the translational start. The minor transcript, with a promoter region sharing homology with a consensus nif promoter of Klebsiella pneumoniae and also with other Pseudomonas genes, is initiated 145 base pairs upstreams of the azurin initiation codon. Downstream from the azurin structural gene a sequence similar to a transcriptional terminator is found. Northern blot analysis indicated two sizes of the azurin mRNA (0.54 kb and 0.65 kb) confirming the S1 mapping and the predictions from the nucleotide sequence.