Gel Native PAGE Research Articles

Identification of pheromone-binding proteins of the maize stem borer, Chilo partellus (Swinhoe, 1885) (Lepidoptera: Crambidae)

Pheromone-binding proteins (PBPs) play a significant role in olfaction and mating. The present work was designed to isolate, extract, and purify the pheromone-binding proteins from the antennae of male Chilo partellus (Swinhoe, 1885) (Lepidoptera: Crambidae). The pheromone-binding proteins extracted from the male antennae were found to be 770 μg in 100 mg of sample. Pheromone-binding protein molecular weight was determined as 17 kDa by SDS-PAGE analysis. Identified proteins were purified through gel extraction with a recovery percentage of proteins up to 95%. Purified protein samples are analyzed on native PAGE gels. Relative mobility of proteins was determined as 0.574 nm in the densitometry analysis. These identified pheromone-binding proteins can be used for identification of novel pheromone compounds in in vitro studies. This study can be helpful in designing integrated pest management programs to control the maize stem borer by mass trapping of male moths.

A comparative analysis of human plasma and serum proteins by combining native PAGE, whole-gel slicing and quantitative LC-MS/MS: Utilizing native MS-electropherograms in proteomic analysis for discovering structure and interaction-correlated differences.

MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.

MHC II binds to multiple sites on H1N1 hemagglutinin protein

Abstract The exact mechanisms of antigen processing within an antigen presenting cell (APC) are not well defined. The canonical pathway describes degradation of the antigenic proteins, competition between peptides for binding to MHC II, followed by presentation to T cells. Alternately, an entire protein might bind to MHC II first and be trimmed through the action of enzymes. Here we determined the binding affinity of HA to DR1, a human MHC II allele, in the presence and absence of the peptide-editing molecule DM and whether multiple DR1 can bind to a single HA. Binding affinities were determined by competitive binding assays with or without DM. Both native and denatured HA bind to DR1 with comparable affinities in the hundreds of nanomolar range. DM appears to decrease binding strength of HA irrespective of its state. Multiple binding events on individual HA were shown over time on blue native-PAGE gels. Bands of increasing molecular weight appear over a 24-hour time course in the blue native gels while amounts of HA and DR1 decrease over time. Taken together, these results support the hypothesis of an alternate pathway of antigen processing whereby the protein binds first to potentially multiple MHC II molecules and is then trimmed by enzymatic activity. Such MHC-mediated shielding mechanism may increase presentation of peptides by APCs that would be otherwise lost to cleavage.

Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections.

Neutrophils have an important role in the antimicrobial defense and resolution of urinary tract infections (UTIs). Our research suggests that a mechanism known as neutrophil extracellular trap (NET) formation is a defense strategy to combat pathogens that have invaded the urinary tract. A set of human urine specimens with very high neutrophil counts had microscopic evidence of cellular aggregation and lysis. Deoxyribonuclease I (DNase) treatment resulted in disaggregation of such structures, release of DNA fragments and a proteome enriched in histones and azurophilic granule effectors whose quantitative composition was similar to that of previously described in vitro-formed NETs. The effector proteins were further enriched in DNA-protein complexes isolated in native PAGE gels. Immunofluorescence microscopy revealed a flattened morphology of neutrophils associated with decondensed chromatin, remnants of granules in the cell periphery, and myeloperoxidase co-localized with extracellular DNA, features consistent with early-phase NETs. Nuclear staining revealed that a considerable fraction of bacterial cells in these structures were dead. The proteomes of two pathogens, Staphylococcus aureus and Escherichia coli, were indicative of adaptive responses to early-phase NETs, specifically the release of virulence factors and arrest of ribosomal protein synthesis. Finally, we discovered patterns of proteolysis consistent with widespread cleavage of proteins by neutrophil elastase, proteinase 3 and cathepsin G and evidence of citrullination in many nuclear proteins.

The Detection of a GABAAR β3 Subunit in an Affinity-Purified Preparation of the GABAAR-Associated Cl-/HCO3 --ATPase Isolated from Rat Brain

Background: The Cl-/HCO3--ATPase from animal brain is a new primary active Cl-- transport system that is coupled to the GABAA receptor (GABAAR). The structural coupling of the enzyme with GABAAR, however, has not been confirmed by the presence of GABAAR subunits within the purified protein complex. Objective/Design: Identification of the enzyme using methodological approaches that would allow the demonstration of GABAAR peptide subunits within a purified enzyme preparation. Main outcome measures: A plasma membrane-enriched preparation (PMP) and a purified enzyme preparation (PEP) from rat brain were loaded onto high resolution clear native-PAGE (hrCNE-PAGE) gels and analyzed on a denaturing gel system. The Cl-/HCO3--ATPase activity was detected in gel strips in the presence of Mg2+-ATP and 5 mM Cl-/25 mM HCO3-, and was inhibited by ethanol (500 mM). The enzyme in the gel strips was detected by western blotting with antibodies against GABAAR 1-3 subunit. Results: The PEP gave one band with a molecular weight of 300 kDa. HrCNEPAGE/ SDS and western blotting results confirmed the presence of the GABAAR β 1-3 subunits in the PMP. The PEP showed two subunits with molecular masses of 52 kDa and 57 kDa that were immuno-reactive only with GABAAR 3 antibodies. These peptide sub-units were directly phosphorylated by γ-32 P-ATP and dephosphorylated in the presence of Cl-/HCO3-, hydroxylamine and ethanol. Conclusion: This proteomic approach and western blot analysis allowed the detection of the Cl-/HCO3--АTPase along with GABAAR 3 subunits isolated from rat brain.

Subunit CcoQ is involved in the assembly of the Cbb3-type cytochrome c oxidases from Pseudomonas stutzeri ZoBell but not required for their activity

The Cbb3-type cytochrome c oxidases (Cbb3-CcOs), the second most abundant CcOs, catalyze the reduction of molecular oxygen to water, even at micromolar oxygen concentrations. In Pseudomonas stutzeri ZoBell, two tandemly organized cbb3-operons encode the isoforms Cbb3-1 and Cbb3-2 both possessing subunits CcoN, CcoO and CcoP. However, only the cbb3-2 operon contains an additional ccoQ gene. CcoQ consists of 62 amino acids and is predicted to possess one transmembrane spanning helix. The physiological role of CcoQ was investigated based on a CcoQ-deletion mutant and wild-type Cbb3-2 crystals not containing subunit CcoQ. Cbb3-2 isolated from the deletion mutant is inactive and appears as a dispersed band on blue native-PAGE gels. Surprisingly, in the absence of ccoQ, Cbb3-1 also shows a strongly reduced activity. Our data suggest that CcoQ primarily functions as an assembly factor for Cbb3-2 but is also required for correct assembly of Cbb3-1. In contrast, once correctly assembled, Cbb3-1 and Cbb3-2 possess a full enzymatic activity even in the absence of CcoQ.

A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively

Calpains (CAPN) are a family of Ca2+-dependent cysteine proteases that regulate various cellular functions by cleaving diverse substrates. Of the 15 mammalian calpains, CAPN8 and CAPN9 are two that are expressed predominantly in the gastrointestinal tract, where they interact to form a protease complex, termed G-calpain. However, because native G-calpain exhibits a highly restricted expression pattern, it has never been purified, and the interactions between CAPN8 and CAPN9 have not been characterized. Here, we clarified the molecular nature of G-calpain by using recombinant proteins and transgenic mice expressing FLAG-tagged CAPN8 (CAPN8-FLAG). Recombinant mouse CAPN8 and CAPN9 co-expressed in eukaryotic expression systems exhibited the same mobility as native mouse G-calpain in Blue Native-PAGE gels, and CAPN8-FLAG immunoprecipitation from stomach homogenates of the transgenic mice showed that CAPN9 was the only protein that associated with CAPN8-FLAG. These results indicated that G-calpain is a heterodimer of CAPN8 and CAPN9. In addition, active recombinant G-calpain was expressed and purified using an in vitro translation system, and the purified protease exhibited enzymatic properties that were comparable with that of calpain-2. We found that an active-site mutant of CAPN8, but not CAPN9, compromised G-calpain's substrate cleavage activity, and that the N-terminal helix region of CAPN8 and the C-terminal EF-hands of CAPN8 and CAPN9 were involved in CAPN8/9 dimerization. Furthermore, CAPN8 protein in Capn9-/- mice was almost completely lost, whereas CAPN9 was only partially lost in Capn8-/- mice. Collectively, these results demonstrated that CAPN8 and CAPN9 function as catalytic and chaperone-like subunits, respectively, in G-calpain.

Mechanism of copper uptake from ceruloplasmin by cells and the involvement of an additional copper uptake transporter

Ceruloplasmin (Cp) is the most abundant copper‐containing protein in the blood plasma. Though well known for its ferroxidase activity, it also has other functions. We have now demonstrated it is a circulating source of copper for cells, using 64Cu‐labeled Cp purified from mouse plasma, and mouse embryonic fibroblasts (MEFs) that do and do not express the only as yet identified copper uptake transporter in mammalian cells, CTR1. During copper delivery, Cp was converted from the holo (Cu‐containing) to the apo form, as demonstrated by immunoblotting of native PAGE gels, and 64Cu‐uptake was not reduced by inhibitors of endocytosis. Uptake of Cp‐Cu was significantly greater in cells expressing vs not expressing Ctr1, indicating that Ctr1 can take up copper from Cp. However, most of the uptake was through an as yet unidentified transporter, that like Ctr1 preferred Cu(I) and was inhibited by Ag(I). Attempts to cross‐link Cp to this transporter in the Ctr1−/− MEFs did not result in the identification of plausible transporter proteins. To improve chances of cross‐linking sufficient protein for identification by MS‐MS, studies with plasma membrane fractions from brains and hearts of rats incubated at 4ºC with purified 67Cu‐labeled rat plasma Cp were initiated (a method used previously to identify Cp “receptors”; BBRC 139: 822, 1986) and Cp‐binding to the membranes was confirmed. We conclude that uptake of copper from Cp occurs at the cell surface, with formation of apoCp and delivery of the copper to Ctr1 and an additional transporter.Support or Funding InformationR15 GM100464 from the National Institutes of Health

Modulation of antioxidant enzymes by salicylic acid in arsenic exposed Glycine max L.

To investigate the physiological and metabolic attributes of arsenic (As) stress tolerance conferred by exogenous salicylic acid (SA), Glycine max L. (variety JS 335) seeds were aseptically germinated over filter paper moistened with SA (500 µM) and/or10 and 100 µM As (Sodium arsenite was used). On 2nd and 5th days of germination, the growing radicles were harvested, and analyzed for growth and different metabolic attributes. Findings exemplified that As significantly decreased germination percentage, radicle length, dry mass and activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), while stimulated the contents of As, reactive oxygen species (ROS), lipoxygenase (LOX), guaiacol peroxidase (POD) and proline. Additionally, isozymes of antioxidants were also scrutinized over Native-PAGE gels and were found to be altered considerably under As-stress. However, exogenous SA remarkably enhanced germination percentage, growth indices, activities of SOD, CAT and APX, and proline accumulation along with reduced As, ROS and LOX, and restoring POD in As-stressed seedlings. In conclusion, SA confers As-stress tolerance to Glycine max L. by regulating the antioxidant enzymes and proline accumulation thereby reduced As content and ROS production. Further study is intended, particularly at gene level, to understand precise mechanism(s) involved in SA-mediated As-stress tolerance.

Enzymatic properties of chitinase-producing antagonistic bacterium Paenibacillus chitinolyticus with various substrates

Various chitin substrates were used to investigate the properties of enzymes produced from the chitinase-producing bacterium Paenibacillus chitinolyticus MP-306 against phytopathogens. The MP-306 bacterium was incubated in nine culture media [crab shell powder chitin (CRS), chitin-protein complex powder (CPC), carboxymethyl-chitin powder (CMC), yeast extract only (YE), LB (Trypton, NaCl, and yeast extract), GT (Trypton, NaCl, and glucose), crab shell colloidal chitin (CSC), squid pen powder chitin (SPC), and cicada slough powder chitin (CSP)] at 30 °C for 3 days. Chitinase isozymes in CPC medium were expressed strongly as CN1, CN2, CN3, CN4, CN5, and CN6 bands on native-PAGE gels. Chitinase isozymes in CPC and CMC medium were expressed as 13 bands (CS1–CS13) on SDS-PAGE gels. Chitinase isozymes were expressed strongly on SDS-PAGE gels as two bands (CS6 and CS8) on YE and LB medium and 13 bands (CS1–CS13) on SPC medium. In crude enzyme, chitinase isozymes at pH 7 and pH 9 in chitin media appeared strongly on SDS-PAGE gels. Partial purified enzyme indicated high stability of enzyme activity at various temperatures and pHs in chitin medium, while these enzymes indicated low activity staining of enzyme on electrophoresis gels at various temperatures and pHs condition of chitin medium.

Deletion of nfnAB in Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism.

NfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP(+). The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains of Thermoanaerobacterium saccharolyticum engineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts of T. saccharolyticum. Activity was also detected using activity staining of native PAGE gels. The nfnAB gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum, deletion of nfnAB resulted in a 46% increase in H2 formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss of nfnAB caused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ΔnfnAB strain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ΔnfnAB strain with unchanged ethanol yield had NADH-linked ADH activity. Deletion of nfnAB caused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lost nfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct from nfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity. Thermophilic anaerobes that can convert biomass-derived sugars into ethanol have been investigated as candidates for biofuel formation. Many anaerobes have been genetically engineered to increase biofuel formation; however, key aspects of metabolism remain unknown and poorly understood. One example is the mechanism for ferredoxin oxidation and transfer of electrons to NAD(P)(+). The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP(+) and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. We report the first deletion of nfnAB and demonstrate a role for NfnAB in metabolism and ethanol formation in Thermoanaerobacterium saccharolyticum and show that this may be an important feature among other thermophilic ethanologenic anaerobes.

Blue Native PAGE and Antibody Gel Shift to Assess Bak and Bax Conformation Change and Oligomerization.

Blue native PAGE (BN-PAGE) uses Coomassie dye rather than denaturing SDS to provide a negative charge to proteins for electrophoresis. As such, it is a useful assay for investigating native supramolecular membrane complexes without the need for cross-linking. As Bak and Bax oligomers form in the mitochondrial outer membrane, and they can be efficiently monitored by BN-PAGE. Furthermore, BN-PAGE performed in conjunction with gel-shift using conformation-specific antibodies can provide additional information regarding the activation state of Bak or Bax in specific membrane complexes.

Isolation of a novel lectin from the dorsal spines of the devil stinger, Inimicus japonicus

A novel lectin was purified from the dorsal spines of the devil stinger, Inimicus japonicus using a combination of affinity chromatography techniques. A single band was detected on a native PAGE gel with a relative molecular mass of 97 kDa. The N-terminal partial amino acid of the intact 75 kDa subunit of the 97 kDa lectin was found to be DHEDS. The agglutination of rabbit erythrocytes by the 97 kDa lectin was inhibited most effectively by methyl α-D-mannoside. The 97 kDa lectin stimulated mitogenesis in murine splenocytes. This is the first study to examine the dorsal lectin of I. japonicus and one of the very few studies on venom lectins from venomous scorpaeniform fish. These results suggest that the devil stinger, I. japonicus, may be a novel resource for biologically active substances.

Identifying the Structure‐Function Activity of Human Cytosolic Sulfotransferase (SULT) 1C4

Human cytosolic sulfotransferases (hSULTs) are phase II conjugation enzymes that play a critical role in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. SULTs transfer a sulfuryl‐moiety from the obligate donor PAPS (3′‐phosphoadenosine 5′‐phosphosulfate) to a suitable hydroxyl or amine group of the substrate. While some of the SULTs have been studied extensively, others, such as the SULT 1C family, remain poorly characterized. The objective of this study is to determine the role of hSULT1C4 in drug metabolism by identifying novel substrates and defining the substrate binding, selectivity, and activity of the SULT1C4 enzyme using molecular modeling and kinetic analysis. SULT1C4 was bacterially expressed and purified for in vitro kinetic analysis. Steady‐state kinetic parameters for 1‐naphthol were analyzed using four concentrations of 1‐naphthol (0.5, 1, 2.5, and 7.5 uM) and four concentrations of PAPS (0.5, 1.5, 5, 15 uM). The Km of 1‐naphthol was approximately 1 uM, whereas the data for PAPS suggests the existence of two Kms (1 uM and 12 uM). To determine if these two Kms are due to SULT1C4's dimeric protein state, two different dimerization domain mutants, SULT1C4‐K272A and SULT1C4‐V277E, have been generated. Native page gel analysis has shown that these mutations prevent dimerization resulting in monomeric SULT1C4 proteins. Our data suggests that the dimeric state of SULT1C4 influences PAPS’ ability to bind to the enzyme.Funding sources: NIH ES022606 and GM038953

Preferential expression of a bromoperoxidase in sporophytes of a red alga, Pyropia yezoensis.

A 2,158bp cDNA (PyBPO1) encoding a bromoperoxidase (BPO) of 625 amino acids was isolated from Pyropia yezoensis. Phylogenetic analysis using amino acid sequences of BPOs suggested that P. yezoensis and cyanobacteria were grouped in the same clade and separated from brown algae. Genomic Southern blot analysis suggested that PyBPO1 existed as a single copy per haploid genome. RT-PCR revealed that PyBPO1 was actively expressed in filamentous sporophytes but repressed in leafy gametophytes under normal growth conditions. High expression levels of PyBPO1 in sporophytes were observed when sporophytes were grown under gametophyte conditions, suggesting that preferential expression of PyBPO1 occurs during the sporophyte phase. BPO activity of cell-free extracts from sporophytes and gametophytes was examined by activity staining on native PAGE gel using o-dianisidine. One activity band was detected in sporophyte sample, but not in gametophyte sample. In addition, we found that bromide and iodide were effective substrate, but chloride was not. BPO activity was observed-likely in chloroplasts-when sporophyte cells were incubated with o-dianisidine and hydrogen peroxide. Cellular BPO staining showed the same halogen preference identified by in-gel BPO staining. Based on GS-MS analysis, bromoform was detected in medium containing sporophytes. Bromoform was not detected under dark culture conditions but was detected in the culture exposed to low light intensity (5μmolm(-2)s(-1)) and increased under a moderate light intensity (30μmolm(-2)s(-1)).

Molecular cloning and biochemical characterization of iron superoxide dismutase from the rodent malaria parasite Plasmodium vinckei

Plasmodium parasite utilizes superoxide dismutase family proteins to limit the toxicity of reactive oxygen species, such as produced through hemoglobin degradation. These proteins play an important role in parasite survival during intra-erythrocytic phase. We have identified, and biochemically characterized a putative iron dependent superoxide dismutase from rodent malaria parasite Plasmodium vinckei (PvSOD1). The recombinant PvSOD1 protein was purified to homogeneity through a combination of affinity and gel filtration chromatography. Crosslinking, Native-PAGE and FPLC gel filtration analyses documented that PvSOD1 exists as a dimer in solution, a common feature shared by other Fe-SODs. PvSOD1 is cytosolic in localization and its expression is comparatively higher during trophozoite as compared to that of ring and schizont stages. Enzymatic activity of recombinant PvSOD1 was validated using conventional zymogram analyses and xanthine–xanthine oxidase system. Under optimal conditions, PvSOD1 was highly active and catalyzed the dismutation of superoxide radicals. Furthermore, PvSOD1 showed activity over a broad range of pH and temperature. Inhibition studies suggested that PvSOD1 was inactivated by hydrogen peroxide, and peroxynitrite, but not by cyanide and azide. Since, PvSOD1 plays a central role in oxidative defense mechanism, therefore, characterization of PvSOD1 will be exploited in the screening of new superoxide dismutase inhibitors for their antimalarial activity.

Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation.

Biotin-dependent human acetyl-CoA carboxylases (ACCs) are integral in homeostatic lipid metabolism. By securing posttranslational biotinylation, ACCs perform coordinated catalytic functions allosterically regulated by phosphorylation/dephosphorylation and citrate. The production of authentic recombinant ACCs is heeded to provide a reliable tool for molecular studies and drug discovery. Here, we examined whether the human ACC2 (hACC2), an isoform of ACC produced using the silkworm BmNPV bacmid system, is equipped with proper posttranslational modifications to carry out catalytic functions as the silkworm harbors an inherent posttranslational modification machinery. Purified hACC2 possessed genuine biotinylation capacity probed by biotin-specific streptavidin and biotin antibodies. In addition, phosphorylated hACC2 displayed limited catalytic activity whereas dephosphorylated hACC2 revealed an enhanced enzymatic activity. Moreover, hACC2 polymerization, analyzed by native page gel analysis and atomic force microscopy imaging, was allosterically regulated by citrate and the phosphorylation/dephosphorylation modulated citrate-induced hACC2 polymerization process. Thus, the silkworm BmNPV bacmid system provides a reliable eukaryotic protein production platform for structural and functional analysis and therapeutic drug discovery applications implementing suitable posttranslational biotinylation and phosphorylation.

Dengue envelope domain III-peptide binding analysis via tryptophan fluorescence quenching assay.

In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.

Extracellular l-Asparaginase from a Protease-Deficient Bacillus aryabhattai ITBHU02: Purification, Biochemical Characterization, and Evaluation of Antineoplastic Activity In Vitro

An extracellular L-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg(-1). The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified L-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the K m, V max, and k cat values of the enzyme were 0.257 mM, 1.537 U μg(-1), and 993.93 s(-1), respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α + β class of proteins with approximately 74 % α-helices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-K-K-I-A of purified L-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the L-asparaginase has significant antineoplastic properties.

Preparation and characterization of DNA nanotube based on DNA tile self-assembly nanotechnology

DNA molecular self-assembly technology can be used to build different nanostructures from one dimension to three dimension, and these structures have many potential uses in micro/nano-electronics, nano-biology as well as other fields. We have successfully assembled one dimension DNA nanotubes by using double-crossover (DX) DNA tiles. The nanotube structures were characterized by atomic force microscope (AFM), native-PAGE gel electrophoresis, fluorescence microscope and transmission electron microscope (TEM). The results show that the diameter of these DNA nanotubes are between 7 and 20 nm, and the longest diameter can reach to more than 50 μm. To stabilize these nanotubes, each strand was phosphorylated chemically on the 5'end. After assembling the nanotubes, T4 DNA ligase was used to ligate the nicks. AFM detection indicates that the ligated DNA nanotubes can keep intact tubular structure better than the unligated one, which reveals that the ligation method can strengthen the DNA nanotubes. The robust nanostructures would promote the research of DNA nanotubes in applications of micro/nano-technology.