Over-expression, purification and functional characterization of Celosia ClpS as a fused protein in Escherichia coli

A ClpS homologue from Celosia cristata was expressed as maltose-binding fusion protein under the control of strong inducible tac promoter of pMALc2X vector in TB 1 strain of Escherichia coli. SDS-PAGE analysis showed that fused ClpS is produced as about 63 kDa protein in recombinant bacteria. Expressed product was purified to homogeneity with a yield of about 31 mg/l of bacterial culture. The results indicated that heterologous expression of Celosia ClpS does not affect bacterial growth under different induced conditions. Total cellular antioxidant assessment results revealed that the induction of ClpS activates the bacterial antioxidative system. Since, the purified ClpS did not exhibit antioxidant activity in vitro, we speculated a functional corelation between bacterial protelolytic apparatus and its anti-oxidative system. This prediction may contribute to our better understanding of functional relationship between proteolytic and antioxidative systems in biological worlds in the future investigations.     

Induced expression of <Emphasis Type="Italic">Eco</Emphasis>RI endonuclease as an active maltose-binding fusion protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Among the numerous bacterial Type II restriction enzymes, EcoRI endonuclease is the most extensively studied and is widely used in recombinant DNA technology. Its heterologous overexpression as recombinant protein has already been studied. However, very limited information concerning its fused product is available thus far. In the present study, the EcoRI restriction endonuclease gene was cloned and expressed as a part of maltose-binding fusion protein under the control of strong inducible tac promoter in TB1 strain of Escherichia coli cells. Transformed cells containing pMALc2X-EcoRI recombinant plasmid were unable to grow under experimental conditions. However, fused EcoRI protein was purified (with the yield of 0.01 mg/l of bacterial culture) by affinity chromatography from E. coli cells induced at the late exponential phase of growth. Restriction quality test revealed that the purified product could restrict a control plasmid DNA in vitro.     

Characterization of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> adhesin thiol peroxidase (HP0390) purified from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The antioxidant protein, adhesin thiol peroxidase (HpTpx or HP0390), plays an important role in enabling Helicobacter pylori to survive gastric oxidative stress. The bacterium colonizes the host stomach and produces gastric cancer. However, little information is available about the biochemical characteristics of HpTpx. We expressed recombinant HpTpx in Escherichia coli, purified to homogeneity, and characterized it. The results showed that HpTpx existed in a monomeric hydrodynamic form and the enzyme fully retained its peroxidase and antioxidant activities. The catalytic reaction of the enzyme was similar to an atypical 2-cysteine peroxiredoxin (Prx). The conformation of the enzyme was observed in the presence and absence of dithiothreitol (DTT); similar to other known thiol peroxidases, conformational change was observed in HpTpx by the addition of DTT.     

Molecular analysis of the genus <Emphasis Type="Italic">Anoxybacillus</Emphasis> based on sequence similarity of the genes <Emphasis Type="Italic">recN</Emphasis>, <Emphasis Type="Italic">flaA</Emphasis>, and <Emphasis Type="Italic">ftsY</Emphasis>

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550–600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3–42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Differential responses of lipid peroxidation and antioxidants in <Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis> subjected to drought stress

To evaluate oxidative stress and the plant antioxidant system of Alternanthera philoxeroides [Mart.] Griseb and Oryza sativa L. in the response to drought, root and leaf tissues of drought-treated A. philoxeroides and O. sativa were collected and relative water content, stomatal conductance, the concentrations of malondialdehyde, proline and the activities of superoxide dismutase, peroxidases, catalase and total antioxidative activity investigated. The results showed that drought treatment had almost no effect on relative water content in A. philoxeroides but reduced relative water content in O. sativa. A. philoxeroides maintained a greater stomatal conductance than O. sativa under drought stress. In A. philoxeroides levels of lipid peroxidation were lower than in O. sativa and did not change during the experiment. After exposure to drought, concentrations of proline and activities of superoxide dismutase, peroxidases and catalase in A. philoxeroides were between 10% and 30% higher than in O. sativa, whereas total antioxidative activity in A. philoxeroides was several-fold higher than in O. sativa.     

Photosynthetic characteristics of marine aerobic anoxygenic phototrophic bacteria <Emphasis Type="Italic">Roseobacter</Emphasis> and <Emphasis Type="Italic">Erythrobacter</Emphasis> strains

A coastal Roseobacter strain of marine aerobic anoxygenic phototrophic bacteria (AAnPB) was isolated and phylogenetically determined. The strain OBYS 0001 was characterized by its physiological and biochemical properties with reference to the Erythrobacter longus type strain NBRC 14126. When grown in batch cultures, the growth curves of the both strains were similar. Cellular bacteriochlorophyll a concentrations of the strains reached the maxima in the stationary growth conditions. In vivo fluorescence excitation/optical density spectra between 470 and 600 nm for OBYS 0001 represented higher values than NBRC 14126. Variable fluorescence measurements revealed that the functional absorption cross section (σ) of the bacterial photosynthetic complexes for OBYS 0001 was significantly higher than that for NBRC 14126 under green excitation. These results suggest that Roseobacter can capture green light more efficiently than Erythrobacter for photosynthesis. The photochemical quantum efficiencies (F _v/F _m) of the bacterial photosynthetic complexes for OBYS 0001 were consistently lower than those for NBRC 14126. A relationship between the growth rate and F _v/F _m was significant for OBYS 0001, but that was not found for NBRC 14126. These results suggested that F _v/F _m for AAnPB could not be used as a proxy of the growth rate which is consistent with their mostly heterotrophic characters.     

Expression of a glutathione reductase from <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> enhanced cellular redox homeostasis by modulating antioxidant proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semiquantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to H₂O₂, menadione, and heavy metal (CdCl₂, ZnCl₂ and AlCl₂)-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to H₂O₂ stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.     

Changing the N-terminal sequence protects recombinant <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> circumsporozoite protein from degradation in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Proteolytic degradation is the primary obstacle in the use of the yeast Pichia pastoris for the expression of recombinant proteins. During the production of a recombinant Plasmodium falciparum circumsporozoite protein in this system, the (NANP) _n repeats region at the N-terminus were completely proteolytically degraded. To remove the potential proteolytic site within the recombinant protein, different strategies were tried, including adjusting the cultivation conditions and mutating the sequence at the junction of the repeat domain and C-terminal region, but the degradation continued. However, modification of the N-terminal sequence by adding an epitope-based peptide to the N-terminus not only protected the repeat domain from cleavage by native proteases during longer induction in the yeast host and purification process, but also stabilized this recombinant protein emulsified with adjuvant ISA720 for at least 6 months. The results showed that proteolytic degradation of the recombinant circumsporozoite protein produced in P. pastoris was amino acid sequence (NANP)-specific, and that this effect was likely dependent on the conformation of the recombinant protein.     

Determination of antioxidant activity of various extracts of <Emphasis Type="Italic">Parmelia saxatilis</Emphasis>

The work was conducted with the purpose to evaluate antioxidant activity of Parmelia saxatilis (PS) by different analytical methods. Water and methanol were used as solvents and antioxidative effects were measured by a ferric thiocyanate method (FTC) and thiobarbituric acid test (TBA). The antioxidant activity increased with the increasing amount of extracts (from 50 to 250 μg) added to linoleic acid emulsion. The methanol extract of PS exhibited high antioxidative activity that was not significantly (P < 0.05) different from α-tocopherol, while aqueous extracts of PS showed low antioxidative activity. Similar trends of antioxidant activity were observed using either the FTC or TBA methods. Antioxidant activity, reducing power, free radical scavenging (DPPH^·), superoxide anion radical scavenging, metal chelating and hydrogen peroxide scavenging activities of PS extracts showed dose dependence and increased with concentration of PS extract. The results obtained in the present study indicate that the PS might be a potential source of natural antioxidant.     

<Emphasis Type="Italic">In vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis> antioxidant effects of three Veronica species

The aim of the present study was to examine the antioxidant activity of three Veronica species (Plantaginaceae). The antioxidant potential of various extracts obtained from aerial flowering parts was evaluated by DPPH-free (1,1-diphenyl-2-picrylhydrazyl-free) radical scavenging activity and ferric-reducing antioxidant power assays. Considerable antioxidant activity was observed in the plant samples (FRAP values ranged from 0.97 to 4.85 mmol Fe²⁺/g, and DPPH IC₅₀ values from 12.58 to 66.34 μg/ml); however, these levels were lower than the activity of the control compound butylated hydroxytoluene (BHT) (FRAP: 10.58 mmol Fe²⁺/g; DPPH IC₅₀: 9.57 μg/ml). Also, the in vivo antioxidant effects were evaluated in several hepatic antioxidant systems in rats (activities of glutathione peroxidase, glutathione reductase, peroxidase, catalase, xanthine oxidase, glutathione content and level of thiobarbituric acid reactive substances) after treatment with different Veronica extracts, or in combination with carbon tetrachloride (CCl₄). Pretreatment with 100 mg/kg b.w. of Veronica extracts inhibited CCl₄-induced liver injury by decreasing TBA-RS level, increasing GSH content, and bringing the activities of CAT and Px to control levels. The present study suggests that the extracts analyzed could protect the liver cells from CCl₄-induced liver damage by their antioxidative effect on hepatocytes.     

Functional identification of the gene <Emphasis Type="Italic">bace16</Emphasis> from nematophagous bacterium <Emphasis Type="Italic">Bacillus nematocida</Emphasis>

Bacillus nematocida is a Gram-positive bacterium capable of killing nematodes. Our recent studies identified an extracellular serine protease Bace16 in B. nematocida as a candidate of pathogenic factor in the infection against nematodes, which displayed a high similarity with the serine protease family subtilisin BPN’, and the MEROPS ID is S08.034. To further confirm the roles that bace16 played in the mechanism of nematocidal pathogenesis, recombinant mature Bace16 (rm-Bace16) was expressed in Escherichia coli strain BL21 using pET-30 vector system. Bioassay experiments demonstrated that the purified recombinant protease had the ability to degrade nematode cuticles and kill nematodes. In addition, a bace16 knockout mutant of B. nematocida constructed by homologous recombination showed considerably lower proteolytic activity and less than 50% nematocidal activity than the wild-type strain. These results confirmed that Bace16 could serve as an important virulence factor during the infectious process. Qiuhong Niu and Xiaowei Huang contributed equally to this work.     

Biochemical characterization of 1-Cys peroxiredoxin from <Emphasis Type="Italic">Antrodia camphorata</Emphasis>

Antrodia camphorata is a unique medicinal mushroom found only in Taiwan. It has been used as a remedy for various diseases in folk medicine. Antrodia camphorata has been shown to exhibit antioxidative effects. Peroxiredoxins play important roles in antioxidation and cell signaling. A gene encoding an antioxidant enzyme, 1-cysteine peroxiredoxin (1-Cys Prx), was identified in an expressed sequence tag database of the A. camphorata and cloned by polymerase chain reaction. The 1-Cys Prx cDNA (837 bp, accession no. AY870325) contains an open reading frame encoding a protein of 223 amino acid residues with calculated molecular mass of 25,081 Da. The deduced protein shared 44–58% identity with 1-Cys Prx from Homo sapiens, Bos taurus, and Saccharomyces cerevisia. The sequence surrounding the conserved cysteine DFTPVCTTE is conserved. The coding sequence was subcloned into a vector, pET-20b (+), and transformed into Escherichia coli. The recombinant 1-Cys Prx was purified by Ni²⁺-nitrilotriacetic acid (Sepharose). The purified enzyme was characterized under various conditions. The enzyme is thermostable because its half-life of inactivation was 15.5 min at 60°C. It was stable under alkaline pH range from 7.8 to 10.2. The enzyme showed decreased activity with increasing concentration of imidazole. The enzyme is sensitive to trypsin and chymotrypsin treatment. Lisa Wen, Hui-Ming Huang, and Rong-Huay Juang contributed equally to this paper.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a susceptible host plant for the holoparasite <Emphasis Type="Italic">Cuscuta </Emphasis>spec

Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A. thaliana covering the entire range of its genetic diversity, as well as Arabidopsis halleri, were found to be susceptible towards Cuscuta reflexa.     

Extract from <Emphasis Type="Italic">Conyza canadensis</Emphasis> as a modulator of plasma protein oxidation induced by peroxynitrite <Emphasis Type="Italic">in vitro</Emphasis>

The antioxidative activity of the extract from Conyza canadensis in plasma treated with peroxynitrite (ONOO⁻) (0.1 mM) was studied. C. canadensis is known to possess a broad set of pharmacological effects because of content of various antioxidants, antiplatelet and anticoagulant compounds. The aim of our study was to assess if this extract protects plasma proteins against oxidative/nitrative damages induced by ONOO⁻. The plasma components are continuously exposed to reactive oxygen/nitrogen species action. Peroxynitrite evokes oxidative stress and induces undesirable effects in biological systems and causes damage to biomolecules. The extract from Conyza (50–2500 mg/ml) caused a dose-dependent reduction of protein nitration by 90%. The oxidation of plasma proteins was diminished by about 75%. ONOO⁻ oxidized the plasma thiol groups and this process was inhibited by tested extract. The level of reduced protein thiols was increased thrice at the lowest concentration of extract (50 mg/ml). The highest concentration of extract decreased twice the level of protein thiols in reduced forms and increased the homocysteine level about 4.5 times. The obtained results demonstrated that the extract from Conyza possesses antioxidative properties in vitro, protects plasma proteins against toxicity induced by peroxynitrite and has modulating effects on thiol/disulfide redox status.     

Isolation of a <Emphasis Type="Italic">Latimeria menadoensis</Emphasis><Emphasis Type="Italic">heat shock protein 70</Emphasis> (<Emphasis Type="Italic">Lmhsp70</Emphasis>) that has all the features of an inducible gene and encodes a functional molecular chaperone

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system. K. W. Modisakeng and M. Jiwaji contributed equally to this study.     

Purification and characterization of cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript> from <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

Cytochrome c ₆ , (cyt c ₆) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E _m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.     

Expression of the <Emphasis Type="Italic">yggE</Emphasis> gene protects <Emphasis Type="Italic">Escherichia coli</Emphasis> from potassium tellurite-generated oxidative stress

Potassium tellurite is highly toxic to most forms of life and specific bacterial tellurite defense mechanisms are not fully understood to date. Recent evidence suggests that tellurite would exert its toxic effects, at least in part, through the generation of superoxide anion that occurs concomitantly with intracellular tellurite (Te⁴⁺) reduction to elemental tellurium (Te^o). In this work the putative antioxidant role of YggE from Escherichia coli, a highly conserved protein in several bacterial species and whose function is still a matter of speculation, was studied. When exposed to tellurite, E. coli lacking yggE exhibited increased activity of superoxide dismutase and fumarase C, augmented levels of reactive oxygen species and high concentration of carbonyl groups in proteins. Upon genetic complementation with the homologous yggE gene these values were restored to those observed in the parental, isogenic, wild type strain, suggesting a direct participation of YggE in E. coli tolerance to tellurite.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Cloning and expression of the sucrose phosphorylase gene from <Emphasis Type="Italic">Leuconostoc </Emphasis><Emphasis Type="Italic">mesenteroides</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K _m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.