Cellulose Affinity Chromatography Research Articles

Sequence and phylogenetic analysis of a novel alphaendornavirus, the first virus described from the oomycete plant pathogen Phytophthora heveae

Here, we report the discovery and complete genome sequence of a novel virus, designated as “Phytophthora heveae alphaendornavirus 1” (PhAEV1), from a single isolate of the plant pathogenic oomycete Phytophthora heveae (kingdom Stramenipila) isolated from a tropical evergreen lowland rainforest in northern Vietnam. PhAEV1 was detected by both cellulose affinity chromatography of dsRNA and high-throughput sequencing of total RNA, and its presence and sequence were confirmed by RT-PCR and Sanger sequencing. The PhAEV1 genome, 12,820 nucleotides (nt) in length, was predicted to encode a single large polyprotein with the catalytic core domain of viral (superfamily 1) RNA helicase (HEL, amino acid [aa] positions 1,287-1,531), glycosyltransferase (GT, aa positions ca. 2,800-3,125), and RNA-directed RNA polymerase (RdRp, aa positions 3,875-4,112). PhAEV1 is the most similar to Phytophthora cactorum alphaendornavirus 3, sharing 39.4% and 39.1% nt and aa sequence identity, respectively. In addition to the first 5′-terminal AUG codon, three additional in-frame methionine codons were found in close proximity (nt 14-16, 96-98, and 176-178), suggesting potential additional translation initiation sites. Conserved RdRp motifs (A-E) similar to those detected in related endornaviruses were identified in PhAEV1, as well as in several previously described alphaendornaviruses from other Phytophthora species in which these motifs had not been identified previously. Phylogenetic analysis showed that PhAEV1 clusters with members of the genus Alphaendornavirus in the family Endornaviridae and is basal to two other alphaendornaviruses described from another oomycete, Phytophthora cactorum. PhAEV1 is the first virus reported in P. heveae.

Construction of a Novel Chimeric Dextransucrase Fused to the Carbohydrate-Binding Module CBM2a

Lactic acid bacteria (LAB) have the potential to produce homoexopolysaccharides (HoPS). Their health benefits and physicochemical properties have been the subject of extensive research. The HoPS functional properties are determined by molecular weight, the type of glycosidic linkages, degrees of branching and chemical composition. The dextransucrases (DSases) produce a kind of HoPS (dextrans), which are among the first biopolymers produced at industrial scale with applications in medicine and biotechnology. The glycodiversification opens additional applications for DSases. Therefore, the design and characterization of new DSases is of prime importance. Previously, we described the isolation and characterization of a novel extracellular dextransucrase (DSR-F) encoding gene. In this study, from DSR-F, we design a novel chimeric dextransucrase DSR-F-∆SP-∆GBD-CBM2a, where DSR-F-∆SP-∆GBD (APY repeats and a CW repeat deleted) was fused to the carbohydrate-binding module (CBM2a) of the β-1-4 exoglucanase/xylanase Cex (Xyn10A) of Cellulomonas fimi ATCC 484. This dextransucrase variant is active and the specificity is not altered. The DSR-F-∆SP-∆GBD-CBM2a was purified by cellulose affinity chromatography for the first time. This research showed that hybrids and chimeric biocatalyst DSases with novel binding capacity to cellulose can be designed to purify and immobilize using renewable lignocellulosic materials as supports.

The Genomic DNA Immobilization on Microcrystalline Cellulose and its Application to Separate DNA-Binding Proteins from Kumquat (Fortunella margarita Swingle)

A method of immobilizing genomic DNA on microcrystalline cellulose was described to isolate DNA-binding proteins. At first, DNA fragments were prepared by sonication and immobilized on cellulose phase. After the immobilization, DNA duplex formation was done. Using this microcrystalline cellulose affinity chromatography technique, DNA-binding proteins from kumquat (Fortunella margarita Swingle) leaf samples were isolated and then analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS). LC-MS/MS analysis showed that twenty-eight kinds of protein mainly including histones, protein-synthetic proteins and other DNA-binding proteins were identified. The identification list consists with the results in previous research on DNA-binding proteins isolation. It further suggests that the technique developed in this study can be applied to the effective isolation of DNA-binding proteins.

Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1

Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.

In Vitro Selection and Characterization of Cellulose-Binding RNA Aptamers Using isothermal Amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.

Insoluble Aggregates and Protease-resistant Conformers of Prion Protein in Uninfected Human Brains

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.

Effect of sulfur-containing compounds onBacilluscellulosome-associated ‘CMCase’ and ‘Avicelase’ activities

The isolation of cellulosomes from clostridial sources has been extensively studied; however, the isolation of cellulosomes from facultative soil anaerobes of the family Bacillaceae is not as well characterized. The Bacillus cellulosome (celluloxylanosome) essentially consists of two complex components: C-I and C-II. This multi-component complex enables Bacillus to degrade a variety of carbonaceous compounds as it is composed of several enzymes, such as cellulases, xylanases and other degradative enzymes. The cellulosomal cellulases from Bacillus megaterium were purified using cellulose affinity chromatography, followed by Sepharose 4B gel filtration chromatography. The objective of this investigation was to establish the effect of sulfate and sulfide on cellulosomal 'cellulase' activity. An increase in sulfide concentration led to a general enhancement of cellulosomal-associated cellulolytic activity, whereas an increase in sulfate concentration resulted in an inhibition of the cellulosome-associated cellulolytic activity.

Flavopiridol disrupts STAT3/DNA interactions, attenuates STAT3-directed transcription, and combines with the Jak kinase inhibitor AG490 to achieve cytotoxic synergy

Up-regulated signal transducers and activators of transcription (STAT)-mediated signaling is believed to contribute to the pathogenesis of a variety of solid and hematologic cancers. Consequently, inhibition of STAT-mediated signaling has recently been proposed as a potential new therapeutic approach to the treatment of cancers. Having shown previously that the pan-cyclin-dependent kinase inhibitor flavopiridol binds to DNA and seems to kill cancer cells via that process in some circumstances, we evaluated the hypothesis that flavopiridol might consequently disrupt STAT3/DNA interactions, attenuate STAT3-directed transcription, and down-regulate STAT3 downstream polypeptides, including the antiapoptotic polypeptide Mcl-1. SDS-PAGE/immunoblotting and reverse transcription-PCR were used to assess RNA and polypeptide levels, respectively. DNA cellulose affinity chromatography and a nuclear elution assay were used to evaluate the ability of flavopiridol to disrupt STAT3/DNA interactions. A STAT3 luciferase reporter assay was used to examine the ability of flavopiridol to attenuate STAT3-directed transcription. Colony-forming assays were used to assess cytotoxic synergy between flavopiridol and AG490. Flavopiridol was found to (a) disrupt STAT3/DNA interactions (DNA cellulose affinity chromatography and nuclear elution assay), (b) attenuate STAT3-directed transcription (STAT3 luciferase reporter assay), and (c) down-regulate the STAT3 downstream antiapoptotic polypeptide Mcl-1 at the transcriptional level (reverse transcription-PCR and SDS-PAGE/immunoblotting). Furthermore, flavopiridol, but not the microtubule inhibitor paclitaxel, could be combined with the STAT3 pathway inhibitor AG490 to achieve cytotoxic synergy in A549 human non-small cell lung cancer cells. Collectively, these data suggest that flavopiridol can attenuate STAT3-directed transcription in a targeted fashion and may therefore be exploitable clinically in the development of chemotherapy regimens combining flavopiridol and other inhibitors of STAT3 signaling pathways.

Proteomic Characterization of Messenger Ribonucleoprotein Complexes Bound to Nontranslated or Translated Poly(A) mRNAs in the Rat Cerebral Cortex

Receptor-triggered control of local postsynaptic protein synthesis plays a crucial role for enabling long lasting changes in synaptic functions, but signaling pathways that link receptor stimulation with translational control remain poorly known. Among the putative regulatory factors are mRNA-binding proteins (messenger ribonucleoprotein, mRNP), which control the fate of cytosolic localized mRNAs. Based on the assumption that a subset of mRNA is maintained in an inactive state, mRNP-mRNA complexes were separated into polysome-bound (translated) and polysome-free (nontranslated) fractions by sucrose density centrifugation. Poly(A) mRNA-mRNP complexes were purified from a postmitochondrial extract of rat cerebral cortex by oligo(dT)-cellulose affinity chromatography. The mRNA processing proteins were characterized, from solution, by a nanoflow reverse phase-high pressure liquid chromatography-mu-electrospray ionization mass spectrometry. The majority of detected mRNA-binding proteins was found in both fractions. However, a small number of proteins appeared to be fraction-specific. This subset of proteins is by far the most interesting because the proteins are potentially involved in controlling an activity-dependent onset of translation. They include transducer proteins, kinases, and anchor proteins. This study of the mRNP proteome is the first step in allowing future experimentation to characterize individual proteins responsible for mRNA processing and translation in dendrites.

Expression and purification of recombinant Kaposi’s sarcoma-associated herpesvirus DNA polymerase using a Baculovirus vector system

The DNA polymerase (POL) of Kaposi’s sarcoma-associated herpesvirus (KSHV) is essential for viral DNA replication and, thus, may be considered as a viable target for anti-KSHV therapeutics. To produce large quantities of homogeneous and pure POL required for high-throughput screening (HTS) for inhibitors, we generated a recombinant baculovirus vector encoding a hexahistidine (His6)-tagged POL and infected Spodoptera frugiperda Sf-9 insect cells. High expression of recombinant POL (rPOL) was achieved for up to 72 h post-infection. The rPOL was solubilized in lysis buffer containing 0.3% Cymal-5 detergent, purified by metal-chelating and dsDNA–cellulose affinity chromatography, and analyzed by anti-His antibody Western blot and mass spectrometry. The functionality of rPOL was confirmed by its DNA synthesis activity in vitro, which was effectively blocked by the anti-herpetic DNA polymerase inhibitors, foscarnet and cidofovir diphosphate, in a dose-dependent manner. The POL expressed and purified from the recombinant baculovirus-infected insect cells may be useful toward the development of HTS of large chemical libraries to identify novel KSHV DNA polymerase inhibitors.

Interaction of a low mobility group protein, LMG 160, with deoxyribonucleic acid

A fraction of low mobility group (LMG) nonhistone protein designated LMG 160 was isolated from rat liver chromatin by preparative gel electrophoresis and its interaction with DNA was studied using thermal denaturation and DNA–cellulose affinity chromatography techniques. The results showed that LMG 160 with an isoelecteric point of 5–5.5 was bound to DNA and decreased its melting temperature. Increasing ionic strengths decreased this effect. DNA–cellulose affinity chromatography showed the affinity of LMG 160 to double stranded DNA was higher than that to single stranded DNA, since it required 0.6 M NaCl for elution. The results suggest that LMG 160 protein preferentially binds to double stranded DNA destabilizes it and the binding is electrostatic.

Purification and properties of an endo-beta-1,4-glucanase from strawberry and down-regulation of the corresponding gene, cel1.

An endo-beta-1,4-glucanase (EG) was purified from ripe strawberry (Fragaria x ananassa Duch.) fruit using cellulose affinity chromatography. The purified enzyme gave a single protein band of 54 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this protein showed strong homology with the proteins encoded by recently identified EG genes from different strawberry cultivars and from Arabidopsis, pepper and tomato. The enzyme specifically cleaved the beta-1,4-glucosyl linkages of xyloglucan but was unable to hydrolyze those of insoluble cellulose. The pH optimum and Km of the enzyme against the artificial substrate carboxymethylcellulose (CMC) were pH 5.0-7.0 and 1.3 mg ml-1, respectively. To assess the role of the Cell enzyme in fruit softening a cDNA of the corresponding fruit-specific and ripening-enhanced strawberry gene, cell, was used to down-regulate cell gene expression in transgenic strawberry plants. In several primary transformants, cell mRNA was strongly suppressed in ripe fruit. However, the EG activity and firmness of these fruit were indistinguishable from those of control fruit. The expression of a second gene, cel2, encoding a different strawberry EG was unaltered in the fruits of these transformants. The presence of the cel2 transcript in transgenic plants may have prevented the specific down-regulation of cell from revealing its role in fruit softening.

Studies on carboxymethyl cellulase produced by an alkalothermophilic actinomycete

A novel alkalothermophilic actinomycete having optimum growth at pH 9 and 50°C was isolated from self-heating compost from the Barabanki district of Uttar Pradesh, India. Based on its morphology, susceptibility of spores to heat and novobiocin, guanine–cytosine content of chromosomal DNA and cell wall composition, the organism was classified under Thermomonospora. The alkalothermophilic actinomycete produced 23 IU/ml carboxymethyl cellulase (CMCase). The CMCase was purified by fractional ammonium sulphate precipitation followed by cellulose affinity chromatography and Sephacryl S-200 gel filtration. The CMCase had a molecular weight of 38 KD and pI of 4.1. The enzyme exhibited optimum activity at pH 5 and temperature 50°C. The CMCase showed pH stability in the range 7–10. The enzyme retained 100% activity at 50°C for 72 h and had half-lives of 7 and 3 h at 60°C and 70°C, respectively. The CMCase was stable in the presence of commercial detergents such as Ariel, Henko and Surf Excel, indicating its potential as an additive to laundry detergents.

Isolation and characterization of goldfish Y box protein, a germ-cell-specific RNA-binding protein.

Cyclin B is a regulatory subunit of maturation-promoting factor. In goldfish (Carassius auratus) oocytes, cyclin B is synthesized de novo after stimulation by 17alpha,20beta-dihydroxy-4-pregnen-3-one (maturation-inducing hormone). In this study, we examined goldfish oocyte proteins bound to cyclin B mRNA. Using oligo(dT)-cellulose affinity chromatography and northwestern blotting analysis, we identified a 54-kDa cyclin B mRNA-binding protein (p54). Southwestern blotting analysis showed the binding of p54 to the Y box DNA element (CTGATTGGCCAA), suggesting that p54 is a Y box protein in goldfish. We isolated two cDNA clones, GFYP1 and GFYP2, the latter of which encodes a germ-cell-specific Y box protein. An antibody against a GFYP2 protein recognized p54, suggesting that p54 is identical or highly similar to GFYP2 protein. This is also supported by the finding that a recombinant GFYP2 expressed in bacteria bound to both the Y box DNA element and the goldfish cyclin B mRNA synthesized in vitro. These results suggest that p54 is a germ-cell-specific Y box protein and is a potential masking protein of cyclin B mRNA in goldfish oocytes.

Synergism between the cellulosome-integrating protein CipA and endoglucanase CelD of Clostridium thermocellum

The cellulosome-integrating protein CipA of Clostridium thermocellum was produced from a recombinant clone of Escherichia coli and purified by cellulose affinity and anion exchange chromatography. Two active forms of C. thermocellum endoglucanase CelD were tested for binding and hydrolytic activity on Avicel in the presence and in the absence of CipA. One was 68 kDa CelD, which contains an intact dockerin domain. The other was 65 kDa CelD, in which the dockerin domain is partially deleted. CipA promoted quantitative binding of 68 kDa CelD to Avicel and enhanced its Avicelase activity by at least ten-fold. By contrast, CipA had no effect on the activity nor on the cellulose-binding affinity of the truncated 65 kDa form. These results show that interaction between CipA and the catalytic component CelD is needed for the degradation of cellulose and confirm that the interaction is mediated by the dockerin domain of CelD.

The cellulose system of the anaerobic rumen fungus Neocallimastix frontalis: studies on the properties of fractions rich in endo-(1?4)-?-d-glucanase activity

Seven fractions rich in endoglucanase activity were separated from the extracellular cellulase system of the anaerobic rumen fungus Neocallimastix frontalis. The fractions (ES1, ES3, ES2U1, ES2U2, ES2U4, ES2U3C1 and ES2U3C2) were separated from each other and from a fraction that could solubilize crystalline cellulose (the so-called crystalline-cellulose-solubilizing component, CCSC) by the sequential use of differential adsorption on the microcrystalline cellulose Avicel, gel filtration and affinity chromatography on concanavalin-A-Sepharose. The molecular masses of the endoglucanase fractions, when determined by gel filtration, were 64, 30, 61, 113, 17, 38 and 93 kDa respectively. Each enzyme degraded carboxymethylcellulose and was rich in activity to cellulose swollen in phosphoric acid to break the hydrogen bonding: cellobiose, cellotriose and cellotetraose were released in differing proportions. Each fraction showed a characteristic gradient when the capacity of each enzyme to increase the fluidity of a solution of carboxymethylcellulose was plotted against the increase in reducing power of the solution. Although neither endoglucanase fraction, acting in isolation, could degrade crystalline cellulose, three of the fractions (ES1, ES3 and ES2U1) could act synergistically with the CCSC fraction in this regard. Remarkably, the same three fractions also acted in synergism with the cellobiohydrolases (CBH I and CBH II) of the aerobic fungus Penicillium pinophilum in degrading crystalline cellulose, but only when both cellobiohydrolase enzymes were present in the solution along with any one of the three endoglucanases. These observations support the conclusion that the mechanism of action of the cellulase system of N. frontalis in degrading crystalline cellulose may be similar to that operating in the aerobic fungi.

Characterization of the subunits in an apparently homogeneous subpopulation of Clostridium thermocellum cellulosomes

Clostridium thermocellum cellulosomes isolated by cellulose affinity chromatography were fractionated by anion exchange chromatography into apparently homogeneous subpopulations that differed with respect to enzyme activity and subunit composition. One such subpopulation contained predominantly six subunits and was closely similar to the “subcellulosome” described by Kobayashi et al. (Kobayashi, T., Romaniec, M. P. M., Fauth, U., and Demain, A. L., Appl. Environ. Microbiol., 1990, 56, 3040–3046). Avicelase specific activity of this homogeneous subpopulation was slightly higher than that of unfractionated cellulosomes, but the two preparations were similarly affected by Ca 2+, dithiothreitol, and cellobiose. Determination of their N-terminal sequences and enzyme activities has enabled three of the six major subunits of the subpopulation of cellulosomes to be positively identified as known components of the C. thermocellum cellulase complex; the other three subunits did not match up with previously characterized cellulosomal proteins.

Optimization of subtractive hybridization in construction of subtractive cDNA libraries

The efficiency of subtraction, integrity of residual single-stranded cDNA, and efficient recovery of nanogram quantities of double-stranded cDNA are the three most important factors affecting quality of subtractive hybridization reactions prior to subtractive cDNA library construction. Techniques for efficient isolation of single-stranded cDNA, after subtraction, have greatly improved from early protocols based on hydroxylapatite chromatography to phenol-chloroform extraction of biotin-streptavidin-crosslinked polynucleotides or oligo(dA)-cellulose affinity chromatography. Factors affecting mRNA stability at the hybridization step, however, also have consequences that directly affect the complexity of the library and the length of cDNAs recovered. We have optimized the subtractive hybridization step in subtractive cDNA library construction to ensure that single-stranded cDNAs survive hybridization as near to full length as possible. These improvements have enabled successful construction of subtractive cDNA libraries from the nanogram quantities of single-stranded cDNA remaining after extensive liquid hybridization to high calculated C ot values.

Putative steroid-binding receptors and nonreceptor components and testicular activity in the lizard Podarcis sicula sicula.

Labelled testosterone- and oestradiol-binding molecules have been found in the cytosol and nuclei of lizard testes. DNA-cellulose affinity chromatography was used to separate putative sex-steroid-binding receptors (adhering molecules) and nonreceptor components (nonadhering molecules). A putative androgen receptor (Kd: 10(-10) mol l-1; 3-9 fmol g-1 tissue) was found mainly in the nuclei of testicular cells when actively undergoing spermatogenesis. This suggests that, as in higher vertebrates, testosterone is implicated in spermatogenetic step regulation (meiosis and spermiogenesis) in lizard testis. In the cytosol, testosterone-binding molecules (Kd: 10(-9) mol l-1; 384-784 fmol g-1 tissue) with several properties of androgen-binding proteins are present from autumn to spring. The behaviour of these molecules is consistent with the role assigned to androgen-binding proteins as androgen reservoir. A putative oestrogen receptor is present throughout the sexual cycle, except during the culmination phase (breeding). The putative oestrogen receptor may be involved in the regulation of the first spermatogenetic step (spermatogonia multiplication) and in the induction of post-reproductive refractoriness. This phase is present in temperate-zone lizards. These studies show that the evaluation of sex-steroid-binding molecules is useful in considering the relationships between sex hormones and spermatogenetic activity in the testes of lizards.

A One‐Step Procedure for Isolation of Poly(A)+ mRNA from Isolated Brain Capillaries and Endothelial Cells in Culture

The study of the regulation of low-abundance blood-brain barrier (BBB) transcripts either in isolated brain microvessels or in endothelial cells in tissue culture (ECL cells) requires isolation of poly(A)+ mRNA. Therefore, we describe here a single-step method for isolation of poly(A)+ mRNA from brain capillaries or ECL cells using proteinase K/sodium dodecyl sulfate cell lysis and oligo-deoxythymidine cellulose affinity chromatography. The yield of poly(A)+ mRNA was approximately 15-19 micrograms/g of brain or choroid plexus, 14-17 micrograms per batch of isolated capillaries in a single bovine forebrain (190 g), and 6-12 micrograms/10(7) ECL cells. Northern blot analysis showed characteristic and undegraded 2.1- and 1.7-kb actin transcripts in brain capillaries and a 2.1-kb actin mRNA in brain and ECL cells. Northern analysis was also used to quantify the glucose transporter type I transcript, which is very rare in basal ECL cells, and this mRNA was shown to be up-regulated by glucose deprivation. This method represents a significant improvement in the mRNA yield for brain capillaries or cultured endothelial cells compared with the conventional two-step method.