Synthesis and properties of a novel biosuperabsorbent from alkali soluble <Emphasis Type="Italic">Rhizomucor pusillus</Emphasis> proteins

A novel biosuperabsorbent protein hydrogel was prepared from protein-rich alcoholic–alkali soluble parts of zygomycete Rhizomucor pusillus biomass. The fungal protein content was 46.8%, and the lipid content was 13.1%. Extraction of protein from this microorganism through the method applied prevents protein decomposition, resulting in maximum yield. After alcoholic–alkaline extraction, the proteins from the biomass were acylated using ethylenediaminetetraacetic dianhydride and subsequently treated with glutaraldehyde as a crosslinker for further experiments. Thermal consistency was investigated by means of two different methods: thermal denaturation via differential scanning calorimetry and thermal decomposition study via thermogravimetric analysis. The swelling behaviour of the crosslinked hydrogel was measured in deionised water, 0.9% NaCl solution and synthetic urine, which were 87.6, 43 and 38.6 g/g water after 24 h, respectively. Moreover, the isoelectric point (pI) of the hydrogel was determined as pH = 8 by studying swelling behaviour at different pHs. In addition, the dependencies of the swelling behaviour with regard to the chemical modification, the ionic strength, the degree of crosslinking, as well as water absorbency with or without load were studied.     

Construction of an <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> cell-surface display system using a putative GPI-anchored protein

A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of β-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, β-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins.     

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.     

The impact of melaphene on the expression of chloroplastic chaperone protein HSP70B and photosynthetic pigments in cells of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The effects of growth regulator of the new generation—melamine salt of bis(oxymethyl)phosphine acid (melaphene)—on culture growth, pigment and protein content, and the induction of protective chloroplastic chaperone HSP70B in Chlamydomonas reinhardtii CW15 cells were studied. Melaphene exhibited 10–30% growth inhibition at 10⁻⁹–10⁻²% concentration. At 10⁻⁹–10⁻⁴% of melaphene electrophoretic concentration, the pattern of cellular proteins was similar to the control. The alterations in protein content of algae cells were detected only at 10⁻²% concentration. The content of chlorophyll and carotenoids in melaphene-treated cells was 17–40% lower than in the control. Melaphene at 10⁻⁹–10⁻²% concentration inhibited HSP70B induction by 39–43% compared to untreated cells. The potential mechanism of melaphene effect might involve its influence on nuclear gene expression.     

Identification of a glucose-6-phosphate isomerase involved in adaptation to salt stress of <Emphasis Type="Italic">Dunaliella salina</Emphasis>

The unicellular green alga Dunaliella salina is a recognized model for studying plant adaptation to high salinity. To isolate some salt-induced proteins at proteomics levels and to identify their expressions at gene levels, algal cells at logarithmic phase cultured in 1.5 and 3.5 M NaCl media were harvested for protein extraction. Solubilized proteins were applied to two-dimensional gel electrophoresis (2-DE) and analyzed by ImageMaster 2D Platinum software. Twenty-one protein spots whose intensities were elevated threefold to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. One salt-induced protein isolated from the 2-DE gels was identified as a glucose-6-phosphate isomerase (GPI) from D. salina (DsGPI). A full-length cDNA of DsGPI was obtained using rapid amplification of cDNA end technique, and it was shown by heterologous expression to encode a protein with a molecular weight consistent with the protein spot in the 2-DE gels. Real-time quantitative RT-PCR demonstrated that the mRNA of DsGPI was induced up to eightfold (P < 0.01) by 2.5 M and 14-fold higher (P < 0.01) by 3.5 M NaCl than by 1.5 M NaCl, respectively. It is concluded that the protein isolated through 2-DE is indeed DsGPI and that the DsGPI gene may be involved in adaptation to high salinity.     

Production of Human Rotavirus and <Emphasis Type="Italic">Salmonella</Emphasis> Antigens in Plants and Elicitation of fljB-Specific Humoral Responses in Mice

A Nicotiana benthamiana transient expression system was used to express single antigen and dimeric combinations of the human rotavirus (HRV) VP7 and a truncated VP4 (VP4Δ) proteins fused with Salmonella typhimurium’s flagellin fljB subunit. Immunoblot analyses using rabbit antibodies generated against these proteins demonstrated that the constructs were successfully expressed with yields ranging from 0.85 to 31.97 μg of recombinant protein per gram of fresh leaf tissue. Expressing the single and dimeric antigens has no effect on plant growth and development except for VP7 and VP4Δ::VP7, which show mild necrotic lesions. Immunization of mice with proteins from leaves transformed with constructs bearing the fljB moiety elicited an fljB-specific humoral response. The Nicotiana benthamiana transient system is efficient to express multiple combinations of pathogen proteins and demonstrates the potential of generating a Salmonella typhimurium subunit vaccine in plants.     

Obtaining recombinant forms of the outer membrane protein F of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and assessment of their immunogenic properties

Two recombinant forms of the outer membrane protein F (OprF) of Pseudomonas aeruginosa were obtained, the full-length protein OprF and the C-terminal part of the OprF protein (aa 192–342). As a result of double immunizations, these recombinant proteins provided mice with resistance to experimental intraperitoneal challenge with P. aeruginosa. The best protective effects were observed at a dose of 25 μg for OprF and 50 μg for the truncated OprF variant (indices of efficiency were 3.3 and 2.8, respectively). Rabbit antisera immune to the recombinant proteins were also able to protect mice from the experimental infection with P. aeruginosa. Indices of efficiency were 6.4 for OprF and 6.0 for the OprF C-terminal part; these values were approximately two times as high as the effect of sera from intact test animals (3.2).     

A conidial protein (CP15) of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses

Aerial conidia are central dispersing structures for most fungi and represent the infectious propagule for entomopathogenic fungus Beauveria bassiana, thus the active ingredients of commercial mycoinsecticides. Although a number of formic-acid-extractable (FAE) cell wall proteins from conidia have been characterized, the functions of many such proteins remain obscure. We report that a conidial FAE protein, termed CP15, isolated from B. bassiana is related to fungal tolerance to thermal and oxidative stresses. The full-length genomic sequence of CP15 was shown to lack introns, encoding for a 131 amino acid protein (15.0 kDa) with no sequence identity to any known proteins in the NCBI database. The function of this new gene with two genomic copies was examined using the antisense-RNA method. Five transgenic strains displayed various degrees of silenced CP15 expression, resulting in significantly reduced conidial FAE protein profiles. The FAE protein contents of the strains were linearly correlated to the survival indices of their conidia when exposed to 30-min wet stress at 48°C (r ² = 0.93). Under prolonged 75-min heat stress, the median lethal times (LT₅₀s) of their conidia were significantly reduced by 13.6–29.5%. The CP15 silenced strains were also 20–50% less resistant to oxidative stress but were not affected with respect to UV-B or hyperosmotic stress. Our data indicate that discrete conidial proteins may mediate resistance to some abiotic stresses, and that manipulation of such proteins may be a viable approach to enhancing the environmental fitness of B. bassiana for more persisting control of insect pests in warmer climates.     

<Emphasis Type="Italic">Ptcorp</Emphasis> gene induced by cold stress was identified by proteomic analysis in leaves of <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> (L.) Raf.

A proteomic approach was employed to investigate the cold stress-responsive proteins in trifoliate orange (Poncirus trifoliata (L.) Raf.), which is a well-known cold tolerant citrus relative and widely used as rootstock in China. Two-year-old potted seedlings were exposed to freezing temperature (−6°C) for 50 min (nonlethal) and 80 min (lethal), and the total proteins were isolated from leaves of the treated plants. Nine differentially accumulated proteins over 2-fold changes in abundance were identified by two-dimensional gel electrophoresis and mass spectrometry. Among these proteins, a resistance protein induced by the nonlethal cold treatment (protein spot #2 from P. trifoliata) was selected as target sequence for degenerated primer design. By using the designed primers, a PCR product of about 700 bp size was amplified from P. trifoliata genomic DNA, which was further cloned and sequenced. A nucleotide sequence of 676 bp was obtained and named Ptcorp. Blast retrieval showed that Ptcorp shared 88% homology with an EST of cold acclimated Bluecrop (Vaccinium corymbosum) library (Accession number: CF811080), indicating that Ptcorp had association with cold acclimation. Semiquantitative RT-PCR analysis demonstrated that Ptcorp gene was up-regulated by cold stress which was consistent with the former result of protein expression profile. As the resistance protein (NBS-LRR disease resistance protein family) gene was up-regulated by cold stress in trifoliate orange and satsuma mandarin, it may imply that NBS-LRR genes might be associated with cold resistance in citrus.     

Generation of catalytic protein particles in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells using the cellulose-binding domain from <Emphasis Type="Italic">Cellulomonas fimi</Emphasis> as a fusion partner

The cellulose-binding domain (CBD) of a Cellulomonas fimi exo-glucanase was translationally fused with β-glucuronidase (GusA) from Escherichia coli and β-glycosidase (BglA) from Thermus caldophilus, respectively. Two fusion proteins (GusA-CBD and BglA-CBD) were expressed as insoluble aggregates in cells and isolated by centrifugation of the cell lysates. Interestingly, activity assays revealed that > 90% of the catalytic activity of both proteins was localized in the insoluble fractions. For example, the GusA-CBD particles exhibited 21 units per mg protein, which corresponded to 19% specific activity of the highly purified soluble GusA. The specific activity increased further up to 42 units per mg protein when treated with either sonication or chaotropic L-arginine. These results demonstrate that fusion with CBD family II may activate catalytic protein particles in E. coli cells, and that internal proteins of the particles are also active. Finally, the protein particles were tested in repeated batch operations after being cross-linked with chemicals, indicating that they have potential as a new preparation for immobilized biocatalysts.     

Expression of Two Self-enhancing Antifreeze Proteins from the Beetle <Emphasis Type="Italic">Dendroides canadensis</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Antifreeze proteins depress the non-equilibrium freezing point of aqueous solutions, but only have a small effect on the equilibrium melting point. This difference between the freezing and melting points has been termed thermal hysteresis activity (THA). THA identifies the presence and relative activity of antifreeze proteins. Two antifreeze protein cDNAs, dafp-1 and dafp-4, encoding two self-enhancing (have a synergistic effect on THA) antifreeze proteins (DAFPs) from the beetle Dendroides canadensis, were introduced into the genome of Arabidopsis thaliana via Agrobacterium-mediated floral dip transformation. Southern blot analysis indicated multiple insertions of transgenes. Both DAFP-1 and/or DAFP-4 were expressed in transgenic A. thaliana as shown by RT-PCR and Western blot. Apoplastic fluid from T ₃ DAFP-1 + DAFP-4-producing transgenic A. thaliana exhibited THA in the range of 1.2–1.35°C (using the capillary method to determine THA), demonstrating the presence of functioning antifreeze proteins (with signal peptides for extracellular secretion). The freezing temperature of DAFP-1 + DAFP-4-producing transgenic A. thaliana was lowered by approximately 2–3°C compared with the wild type.     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> LHP1 protein is a component of euchromatin

The HP1 family proteins are involved in several aspects of chromatin function and regulation in Drosophila, mammals and the fission yeast. Here we investigate the localization of LHP1, the unique Arabidopsis thaliana HP1 homolog known at present time, to approach its function. A functional LHP1–GFP fusion protein, able to restore the wild-type phenotype in the lhp1 mutant, was used to analyze the subnuclear distribution of LHP1 in both A. thaliana and Nicotiana tabacum. In A. thaliana interphase nuclei, LHP1 was predominantly located outside the heterochromatic chromocenters. No major aberrations were observed in heterochromatin content or chromocenter organization in lhp1 plants. These data indicate that LHP1 is mainly involved in euchromatin organization in A. thaliana. In tobacco BY-2 cells, the LHP1 distribution, although in foci, slightly differed suggesting that LHP1 localization is determined by the underlying genome organization of plant species. Truncated LHP1 proteins expressed in vivo allowed us to determine the function of the different segments in the localization. The in foci distribution is dependent on the presence of the two chromo domains, whereas the hinge region has some nucleolus-targeting properties. Furthermore, like the animal HP1β and HP1γ subtypes, LHP1 dissociates from chromosomes during mitosis. In transgenic plants expressing the LHP1–GFP fusion protein, two major localization patterns were observed according to cell types suggesting that localization evolves with age or differentiation states. Our results show conversed characteristics of the A. thaliana HP1 homolog with the mammal HP1γ isoform, besides specific plant properties.     

Use of SSR,RAPD markers and protein profiles based analysis to differentiate <Emphasis Type="Italic">Eleusine coracana</Emphasis> genotypes differing in their protein content

Fifty-two genotypes of Eleusine coracana collected from Uttarakhand hills were subjected to simple sequence repeat (SSR), random amplified polymorphic DNA (RAPD)-PCR and protein profiling analysis to investigate the variation in protein content. The main objective of the present study was to detect variability among E. coracana and also assess the discriminating ability of these three molecular methods. A total of 21 RAPD and 24 SSR primers were assayed for their specificity in detecting genetic variability in E. coracana, of which 20 RAPD and 21 SSR primers were highly reproducible and were found suitable for use in PCR analysis. Assessing genetic diversity among E. coracana genotypes by RAPD-PCR using 20 polymorphic primers yielded 56 different RAPD markers which clustered the genotypes into different groups on the basis of protein content. Similarly, SSR-PCR with 21 polymorphic primers clustered the genotypes into different groups. On the other hand, biochemical typing of E. coracana using whole seed proteins generated profiles that showed no major difference indicating the technique to be not useful in typing genotypes of this crop. However, a few of the genotypes showed the presence of a unique band of 32 kDa that needs to be further investigated to understand the role of the protein from nutritional point of view, if any. In the present study, significant negative correlation (r = −0.69*) was found between the protein and calcium content of finger millet genotypes. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis based seed storage proteins generated profiles showed no major differences in banding pattern among 52 finger millet genotypes while quantitative estimation of seed storage protein fractions using Lowry method revealed that glutelin was highest followed by prolamin, globulin and albumin.     

Extracellular production of lipoxygenase from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on wheat protein

In this study, the lipoxygenase (ana-LOX) gene from Anabaena sp. PCC 7120 was successful expressed and secreted in Bacillus subtilis. Under the control of the P43 promoter, with a signal peptide from the B. subtilis 168 nprB gene, and facilitated by the molecular chaperone PrsA, the production of the recombinant ana-LOX (ana-rLOX) reached 76 U/mL (171.9 μg/ml) in the supernatant. The purified ana-rLOX was investigated for its effect on dough protein. Ana-rLOX treatment decreased free sulfhydryl groups, increased glutenin macropolymer content, promoted the formation of covalent bonds between gluten protein, and affected protein crosslinking. The results indicated that large aggregates involving gliadin and glutenin were formed. The glutenin macropolymer played a role in the formation of the dough network structure through the exchange of thiol disulfide bonds and the formation of hydrogen or hydrophobic bonds with other proteins.     

Heterologous interferons synthesis in yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed. There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features of its metabolism.     

High-level production of a kringle domain variant by high-cell-density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human kringle domains (KDs) are ubiquitously expressed binding modulators that fold into seven flexible loops and it has been previously demonstrated that KDs can be engineered toward target-specific binding proteins as a non-antibody protein scaffold. Here, we report a method for efficient expression of a KD derivative (KD548)—a promising anti-cancer agent—by high-cell-density culture of Escherichia coli at a preparative scale production. The correct folding of KD548 requires three disulfide bonds. Nevertheless, cytoplasmic expression of KD548 in E. coli led to good yields of highly soluble proteins with high activity. For efficient expression, four sets of expression systems consisting of different promoters (lac or T7) and fusion tags (His or FLAG) were examined. Of these, the expression system using a combination of the T7 promoter with the FLAG tag resulted in the highest production in shake flask cultivation as well as in high-cell-density cultivation performed in a 6.6-L jar bioreactor. When protein expression was induced at high-cell density (optical density [OD] = 100) and when complex feeding solutions were supplemented, cell density (maximum OD = 184) and production yield (∼5.4 g/L) were significantly enhanced to values that were much higher than those found previously with Pichia cultivation (<8 mg/L).     

Proteomic analysis of human skeletal muscle (<Emphasis Type="Italic">m. vastus lateralis</Emphasis>) proteins: Identification of 89 gene expression products

Proteins from bioptates and autoptates of human skeletal muscle m. vastus lateralis were separated by O’Farrell two-dimensional gel electrophoresis (2DE). MALDI-TOF MS and MS/MS enabled identification of 89 protein spots as expression products of 55 genes. A modification of the O’Farrell’s method including non-equilibrium electrophoresis in a pH gradient allowed detection — among major sarcomeric, mitochondrial, and cytosolic proteins — of several proteins, such as PDZ- and LIM domain-containing ones (pI > 8.70), fragments of known proteins, and a stable complex of heavy and light ferritin chains. The data underlie further studies of human skeletal muscle proteins in terms of molecular mechanisms of some physiological and pathological processes.