Native PAGE Analysis Research Articles

Comparative analysis of activities of vital defence enzymes during induction of resistance in pearl millet against downy mildew

Pearl millet [Pennisetum glaucum (L.) R. Br.] has the seventh largest annual production in the world giving it significant economic importance. Although generally well adapted to the growing conditions in arid and semi-arid regions, major constraints to yields are susceptibility to downy mildew disease caused by the oomycete Sclerospora graminicola (Sacc.) Schroet. Induction of resistance against downy mildew disease of pearl millet has been well established using various biotic and abiotic inducers. The present study demonstrated the comparative analysis of the involvement of the important defence enzymes like β-1,3-Glucanase, chitinase, phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and lipoxygenase (LOX) during induced systemic resistance (ISR) mediated by inducers like Benzo(1,2,3)-thiadiazole-7-carbothionic acid-S-methyl ester (BTH), Beta amino butyric acid (BABA), Chitosan and Cerebroside against pearl millet downy mildew disease. Native-PAGE showed six POX isozymes in all categories of uninoculated pearl millet seedlings and maximum intensity of bands was noticed in resistant seedlings. After inoculation in Cerebroside-treated seedlings, there were seven isoforms, POX-4 was not present in any other seedlings. Native-PAGE analysis showed the presence of five PPO isozymes in all categories of uninoculated pearl millet seedlings and after inoculation seven isoforms of PPO-7 were noticed, and the intensity of banding was more in resistant and Cerebroside-treated seedlings. The isoforms PPO-3 were present as an extra band after inoculation in all seedlings. Isoform PPO-7, though found in all seedlings, was very prominent in Chitosan- and Cerebroside-treated seedlings. β-1,3-Glucanase Native-PAGE analysis showed the presence of only one isozyme in all categories of uninoculated/inoculated pearl millet seedlings. Glu-1 isozyme was very prominent in all seedlings including resistant and susceptible seedlings. Among the induced resistant seedlings, highest intensity was observed in Cerebroside-treated seedlings. Native-PAGE analysis showed the presence of three LOX isozymes in all categories of uninoculated pearl millet seedlings, and the intensity of banding pattern was very low in BTH-treated seedlings. LOX-1 and LOX-2 were very prominent in resistant, Chitosan- and Cerebroside-treated seedlings. Upon inoculation, one extra band, LOX-3, was exclusively noticed in Cerebroside-treated seedlings. In inoculated seedlings, LOX-1, LOX-2 and LOX-4 were very prominent in Chitosan Cerebroside-treated seedlings compared to other seedlings.

Reduction of salt-requirement of halophilic nucleoside diphosphate kinase by engineering S–S bond

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S–S bond. It showed increased thermal stability by about 10°C in 0.2M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1M NaCl, while the wild type required 2M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S–S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1M salt. This native-like structure was then converted to the native hexamer with time.

Purification and characterization of an acidothermophilic cellulase enzyme produced by Bacillus subtilis strain LFS3

In the present investigation, a microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated and identified as Bacillus subtilis strain LFS3 by 16S rDNA sequence analysis. The carboxymethylcellulase (CMCase) enzyme produced by the B. subtilis strain LFS3 was purified by (NH₄)₂SO₄ precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 15 %. Native-PAGE analysis of purified CMCase revealed the molecular weight of enzyme to be about 185 kDa. The activity profile of CMCase enzyme showed the optimum activity at temperature 60 °C and pH 4.0, respectively. The enzyme activity was induced by Na⁺, Mg²⁺, NH₄⁺, and EDTA, whereas strongly inhibited by Hg²⁺ and Fe³⁺. The purified enzyme hydrolyzed CMC, filter paper, and xylan, but not p-nitrophenyl β-D-glucopyranoside and cellulose. Kinetic analysis of purified enzyme showed the K(m) value of 2.2 mg/ml. Thus, acidophilic as well as thermophilic nature makes this cellulase a suitable candidate for current mainstream biomass conversion into fuel and other industrial processes.

Isolation and characterization of a new phytoferritin from broad bean (Vicia faba) seed with higher stability compared to pea seed ferritin

Plant ferritin widely occurring in legume seeds represents a novel alternative dietary iron source for treatment of iron deficiency anemia. However, recent studies have shown that known plant ferritins such as SSF and PSF are unstable because they contain a higher amount of the H-1 subunit. Therefore, it is of special interest to explore a new plant ferritin with a lower amount of the H-1. In the present study, a new ferritin (BBSF) was purified from broad bean seeds by consecutive anion exchange and size exclusion chromatography to homogeneity. The apparent molecular mass of BBSF was found to be 560kDa by native-PAGE analysis. N-terminal sequence, MALDI-TOF-MS, and SDS-PAGE analyses indicate that BBSF consists of H-1 and H-2 subunits with a H1/H-2 ratio of 1:6, and shares very high sequence identity with PSF. In contrast, the ratio of H-1 to H-2 in pea seed ferritin (PSF) is 1:2 and in soybean seed ferritin (SSF) is 1:1. Thus, this new protein has quite different subunit composition from PSF and SSF, which might affect its activity in iron uptake. As expected, PSF exhibits a higher catalyzing activity than BBSF, and the association rate of PSF is faster than BBSF upon high iron loading of the protein. More importantly, the stability of BBSF is much higher than that of PSF and SSF under the same experimental conditions, which is beneficial to its application for iron supplement.

α-L-rhamnosidase from Aspergillus clavato-nanicus MTCC-9611 active at alkaline pH

An alpha-L-rhamnosidase secreting fungal strain has been isolated and identified as Aspergillus clavato-nanicus MTCC-9611. The enzyme was purified to homogeneity from the culture filtrate of the fungus using concentration by ultrafiltration membrane and ion-exchange chromatography on CM-cellulose. The native PAGE analysis confirmed the homogeneity of the purified enzyme. The SDS-PAGE analysis of the purified enzyme revealed a single protein band corresponding to the molecular weight 82 kDa. The alpha-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 had optimum at pH 10.0 and 50 degrees C. The K(m) values of the enzyme were 0.65 mM and 0.95 mM using p-nitrophenyl alpha-L-rhamnopyranoside and naringin as a substrates respectively. The enzyme transforms naringin to prunin at pH 10.0 and further hydrolysis of prunin to naringenin does not occur under these reaction conditions that makes alpha-L-rhamnosidase activity of Aspergillus clavatonanicus MTCC-9611 promising enzyme to get prunin for pharmaceutical purposes.

Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

SummaryAn extracellular α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l‐rhamnopyranoside as substrate, Km and Vmax values of the enzyme were 0.30 mm and 27.0 μm min mg−1, respectively. The kcat value was 20.1 s giving kcat/Km value of 67.0 mm s−1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol−1. The α‐l‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l‐rhamnose. The α‐l‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.

Improved biomass saccharification by Trichoderma reesei through heterologous expression of lacA gene from Trametes sp. AH28-2

The Trametes sp. AH28-2 laccase gene lacA fused to cellobiohydrolase I signal peptide coding sequence was heterologously expressed in T. reesei. The lacA cDNA was under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter. Native PAGE analysis indicated that two transformants, L8 and L38, were able to secrete recombinant laccase A, and their laccase activities corresponding to ABTS oxidation reached 3.62 IUml(-1) and 1.50 IUml(-1) respectively. Most of the characteristics of the recombinant laccase were similar to those of the native enzyme. Reducing sugar yields of L8 and L38 obtained from saccharification of corn residue by crude enzyme increased by 31.3% and 71.6% respectively compared to the host strain. These results indicated that the engineering strains developed in this work could be potentially used for laccase production and tailoring cellulase properties with laccase proteins through genetic manipulation would be a feasible strategy to improve saccharification efficiency of biomass by cellulase preparation.

Expression, assembly and function of novel C‐terminal truncated variants of the mouse P2X7 receptor: re‐evaluation of P2X7 knockouts

Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7(-/-) mice (Pfizer KO). Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. RT-PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in-frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS-PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function.

Expression, purification and partial characterization of a xanthine oxidase (XOD) in Arthrobacter sp.

A xanthine oxidase (XOD) was expressed, purified and partially characterized from Arthrobacter sp. with a negative immune protocol. To determine the optimal inducer for XOD, xanthine, hypoxanthine and uric acid were added into the medium of cultivation. The results revealed that with the inducement of about 14mM xanthine, the highest XOD activity could be detected. To separate XOD from Arthrobacter sp., the cells were first cultured without any inducement; then the total proteins of the collected cells were extracted and immunized to rabbits for the polyclonal antibodies. These antibodies were then coupled with sepharose CL 6 B, and the medium was further employed to deplete most of the cells’ back ground proteins. Began with ∼20mg crude protein from disrupted cells was subjected to the antibody medium, and ∼1.45mg protein was detected in unbinding fractions with ∼92.0% of activity. The extracted xanthine oxidase was ∼85% pure with native-PAGE analysis, and ∼90% pure with SDS-PAGE analysis, the yield of protein was ∼7.4%. The specific activity of the enzyme was 36.0U/mg. The native enzyme should be a dimer (∼280kDa) of a protein composed with two different peptides with the mass of approximately 55.5 and 85.5kDa, respectively. The optimal pH and temperature of this enzyme were determined at about pH 7 and 50°C. Furthermore, EDTA revealed almost no influences on the activity.

Degradation of Millimolar Concentration of the Herbicide Dalapon (2,2-Dichloropropionic Acid) by Rhizobium Sp. Isolated from Soil

ABSTRACTThe herbicide Dalapon is widely used in agricultural areas and is persistent in ground water. A Rhizobium sp. was able to grow at 0.2 mM 2,2-dichloropropionic acid (2,2DCP), which was 100-fold lower than the concentration of the substrate routinely used. Apparently, no new dehalogenases are required to allow growth on this low concentration of 2,2DCP as judged by electrophoretic mobility of dehalogenase proteins in native-PAGE analysis and protein separation by anion-exchange column chromatography. The kinetic analysis suggested that the known dehalogenases were able to act efficiently on low concentrations of haloalkanoic acids. The amount of each dehalogenase, from cells grown on low substrate concentration was different compared to that seen at 20 mM 2,2DCP due to complex regulatory controls, which respond to the growth environment.

Decrease of rice plant resistance and induction of hormesis and carboxylesterase titre in brown planthopper, Nilaparvata lugens (Stål) by xenobiotics

The brown planthopper (BPH), Nilaparvata lugens (Stål) is a serious threat to the rice production throughout Asia. The indiscriminate application of various xenobiotics in rice ecosystem is perceived as one of the factors for the frequent outbreak of BPH. The present study has critically analysed the secondary effects of some xenobiotics used in rice field on certain plant and insect parameters that subsequently favour BPH outbreak. Application of 2,4-D, carbendazim, deltamethrin and urea reduced the innate BPH resistance of PTB 33 rice variety due to favourable alterations in rice free amino acid and sucrose content. Similarly, these chemicals also induced hormesis and enhanced feeding in BPH. Alternatively, soil amendment with neem seed powder and Calotropis gigantea leaves improved plant innate resistance and showed no sign of hormesis or enhanced feeding in BPH. In addition, deltamethrin has the ability to stimulate BPH carboxylesterase titre. Native PAGE analysis of esterases from whole body homogenate of BPH revealed at least five esterase isozyme bands, prominent being E1 and E2. However, no difference in BPH esterase banding pattern was observed between different xenobiotic treatments. All these esterase bands are classified under carboxylesterase based on their inhibition by class specific esterase inhibitors.

Stable HIV-1 envelope glycoprotein immune complexes as vaccine immunogens

The development of an HIV-1 vaccine that elicits strong neutralizing antibody (nAb) and T cell responses is challenging. Classical vaccine strategies such as live attenuated vaccines are considered unsafe whereas envelope glycoprotein (Env)subunit vaccines induce low nAb titers that do not protect against HIV-1 infection. We showed previously that most HIV-1-antibody immune complexes (HIV-ICs) formed with either broadly nAbs or Abs derived from patient sera dissociate into free HIV-1 virions and Ab when captured by dendritic cells (DCs). Dissociation of HIV-ICs allows for transmission from DCs to CD4 + T target cells. H but more importantly it can hamper the activation of immune cells which is a hallmark of stable ICs. The natural role of ICs is enhancing uptake by DCs, DC activation, induction of antigen presentation and induction of T cell responses. Furthermore, ICs are captured by follicular DCs that activate the B cells for Ab production, Ab affinity maturation and isotype switching. We explore stable Env-ICs as a vaccine candidate. To form stable Env-ICs we fused the Fc-region of immunoglobulins to trimeric gp140. Env-IC maintained a native Env conformation which was evaluated by ELISA with Env-specific Abs. Native PAGE analyses and size exclusion chromatography showed that Env-ICs formed trimers, but hexamers consisting of 2 Env trimers and 3 dimeric Fc-tails were also observed. The functionality of the Fc-tail was evaluated by immuno-precipitation of the Env-IC with protein-G couple beads. Capture of Env-IC by DCs was enhanced with 50% compared to wild-type Env. Moreover, Env-IC captured by DCs more efficiently activated gp120-specificT helper cells.

Gene cloning, expression, and characterization of a novel acetaldehyde dehydrogenase from Issatchenkia terricola strain XJ-2

Acetaldehyde is a known mutagen and carcinogen. Active aldehyde dehydrogenase (ALDH) represents an important mechanism for acetaldehyde detoxification. A yeast strain XJ-2 isolated from grape samples was found to produce acetaldehyde dehydrogenase with a high activity of 2.28U/mg and identified as Issatchenkia terricola. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD(+) as coenzyme based on the headspace gas chromatography analysis. A novel acetaldehyde dehydrogenase gene (ist-ALD) was cloned by combining SiteFinding-PCR and self-formed adaptor PCR. The ist-ALD gene comprised an open reading frame of 1,578bp and encoded a protein of 525 amino acids. The predicted protein of ist-ALD showed the highest identity (73%) to ALDH from Pichia angusta. The ist-ALD gene was expressed in Escherichia coli, and the gene product (ist-ALDH) presented a productivity of 442.3U/mL cells. The purified ist-ALDH was a homotetramer of 232kDa consisting of 57kDa-subunit according to the SDS-PAGE and native PAGE analysis. Ist-ALDH exhibited the optimal activity at pH9.0 and 40°C, respectively. The activity of ist-ALDH was enhanced by K(+), NH4(+), dithiothreitol, and 2-mercaptoethanol but strongly inhibited by Ag(+), Hg(2+), Cu(2+), and phenylmethyl sulfonylfluoride. In the presence of NAD(+), ist-ALDH could oxidize many aliphatic, aromatic, and heterocyclic aldehydes, preferably acetaldehyde. Kinetic study revealed that ist-ALDH had a k (cat) value of 27.71/s and a k (cat)/K (m) value of 26.80 × 10(3)/(mols) on acetaldehyde, demonstrating ist-ALDH, a catalytically active enzyme by comparing with other ALDHs. These studies indicated that ist-ALDH was a potential enzymatic product for acetaldehyde detoxification.

Direct evidence that RNA inhibits APOBEC3G ssDNA cytidine deaminase activity

APOBEC3G (A3G) is a deoxycytidine deaminase active on ssDNA substrates. In HIV infected cells A3G interacted with reverse transcription complexes where its activity as a deoxycytidine deaminase led to mutation of the viral genome. A3G not only bound ssDNA, but it also had an intrinsic ability to bind RNA. In many cell types that can support HIV replication, A3G ssDNA deaminase activity was suppressed and the enzyme resided in high molecular mass, ribonucleoprotein complexes associated with cytoplasmic P-bodies and stress granules. Using a defined in vitro system, we show that RNA alone was sufficient to suppress A3G deaminase activity and did so in an RNA concentration-dependent manner. RNAs of diverse sequences and as short as 25nt were effective inhibitors. Native PAGE analyses showed that RNA formed ribonucleoprotein complexes with A3G and in so doing prevented ssDNA substrates from binding to A3G. The data provided direct evidence that A3G binding to cellular RNAs constituted a substantial impediment to the enzyme’s ability to interact with ssDNA.

Pharmacological Characterization of Chemically Synthesized Monomeric phi29 pRNA Nanoparticles for Systemic Delivery

Previous studies have shown that the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor folds into a compact structure, constituting a RNA nanoparticle that can be modularized with functional groups as a nanodelivery system. pRNA nanoparticles can also be self-assembled by the bipartite approach without altering folding property. The present study demonstrated that 2'-F-modified pRNA nanoparticles were readily manufactured through this scalable bipartite strategy, featuring total chemical synthesis and permitting diverse functional modularizations. The RNA nanoparticles were chemically and metabolically stable and demonstrated a favorable pharmacokinetic (PK) profile in mice (half-life (T(1/2)): 5-10 hours, clearance (Cl): <0.13 l/kg/hour, volume of distribution (V(d)): 1.2 l/kg). It did not induce an interferon (IFN) response nor did it induce cytokine production in mice. Repeat intravenous administrations in mice up to 30 mg/kg did not result in any toxicity. Fluorescent folate-pRNA nanoparticles efficiently and specifically bound and internalized to folate receptor (FR)-bearing cancer cells in vitro. It also specifically and dose-dependently targeted to FR(+) xenograft tumor in mice with minimal accumulation in normal tissues. This first comprehensive pharmacological study suggests that the pRNA nanoparticle had all the preferred pharmacological features to serve as an efficient nanodelivery platform for broad medical applications.

Functional Stability of Unliganded Envelope Glycoprotein Spikes among Isolates of Human Immunodeficiency Virus Type 1 (HIV-1)

The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T90 values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T90 (p = 0.029), though two ‘outliers’ were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1ADA was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1JR-CSF. Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines.

Evidence for the Involvement of Zinc in the Association of CRISP1 with Rat Sperm During Epididymal Maturation

Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn(2+) present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn(2+), and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn(2+), but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn(2+) concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn(2+) exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn(2+) supported a direct interaction between CRISP1 and Zn(2+). Incubation of either cauda epididymal fluid or purified CRISP1 with Zn(2+), followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn(2+) complexes in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation.

Purification, crystallization and preliminary X-ray analysis of phycocyanin and phycoerythrin from Porphyra yezoensis Ueda.

Porphyra yezoensis is one of the most important and widely cultured seaweeds in China. Phycobiliproteins exhibit excellent spectroscopic properties and play versatile roles in the biomedical, food, cosmetics and chemical synthetic dye industries. Here, the purification and crystallization of phycoerythrin and phycocyanin, two phycobiliproteins extracted from P. yezoensis, are described. Using a novel protocol including co-precipitation with ammonium sulfate and hydroxyapatite column chromatography, both phycobiliproteins were produced on a large scale with improved quality and yield compared with those previously reported. Native PAGE analysis indicated that phycoerythrin and phycocyanin exist as (αβ)(3) heterohexamers in solution. The crystals of phycoerythrin diffracted to 2.07 Å resolution and belonged to space group R3. The unit-cell parameters referred to hexagonal axes are a = b = 187.7, c = 59.7 Å, with nine (αβ)(2) heterotetramers per unit cell. The crystals of phycocyanin diffracted to 2.70 Å resolution in space group P2(1). Matthews coefficient analysis shows that 10-19 (αβ) heterodimers of phycocyanin in the asymmetric unit would be reasonable. A self-rotation function calculation clarified this ambiguity and indicated that 12 (αβ) heterodimers of phycocyanin are assembled in the asymmetric unit.

DcHsp17.7, a small heat shock protein in carrot, is tissue-specifically expressed under salt stress and confers tolerance to salinity

The expression and function of DcHsp17.7, a small heat shock protein in carrot (Daucus carota L.), were examined under salt stress, which is an exacerbating environmental condition due to water shortage and irrigation. DcHsp17.7 was constitutively expressed in leaf and stem tissues under normal growth conditions. Upon exposure to 300 mM NaCl, the protein level of DcHsp17.7 increased dramatically in leaf tissue, but did not significantly change in stem tissue. Native-PAGE analysis showed tissue-specific oligomer formation. Under normal growth conditions, DcHsp17.7 was found in an approximately 240 kDa complex in both tissues. However, NaCl treatment induced an additional approximately 160 kDa complex containing DcHsp17.7. This occurred only in leaf tissue, suggesting tissue-specific oligomeric complex formation. To examine the functional mechanism of DcHsp17.7 under stress conditions, the DcHsp17.7 coding gene was introduced into Escherichia coli. Heterologous expression of DcHsp17.7 was induced by isopropyl β-d-1-thiogalactopyranoside treatment. Upon exposure to salinity, protein levels of DcHsp17.7 decreased, and the protein was not detected after 16 hours. Native-PAGE analysis showed that DcHsp17.7 was present in an approximately 250 kDa complex both before and after salt treatment. Salinity reduced bacterial cell viability; however, the transgenic E. coli expressing DcHsp17.7 exhibited a higher survival rate than control E. coli under salt stress. When the level of soluble proteins was measured under salt stress, transgenic E. coli expressing DcHsp17.7 reproducibly showed slightly higher levels than control cells. This suggests that DcHsp17.7 performs molecular chaperone activity in salt-stressed transgenic E. coli. Our results suggest that DcHsp17.7 is likely to be involved in tolerance not only to thermal stresses but also to other abiotic stresses, such as salinity.

A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

BackgroundClostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology.ResultsA two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation.ConclusionTwo dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes.