Aggregation of Influenza A Virus Nuclear Export Protein

Influenza A virus nuclear export protein (NEP) plays an important role in the viral life cycle. Recombinant NEP proteins containing (His)₆-tag at either N-or C-terminus were obtained by heterologous expression in Escherichia coli cells and their high propensity for aggregation was demonstrated. Dynamic light scattering technique was used to study the kinetics and properties of NEP aggregation in solutions under different conditions (pH, ionic strength, presence of low-molecular-weight additives and organic solvents). Using atomic force microscopy, the predominance of spherical aggregates in all examined NEP preparations was shown, with some amyloid-like structures being observed in the case of NEP-C protein. A number of structure prediction programs were used to identify aggregation-prone regions in the NEP structure. All-atom molecular dynamics simulations indicate a high rate of NEP molecule aggregation and reveal the regions preferentially involved in the intermolecular contacts that are located at the edges of the rod-like protein molecule. Our results suggest that NEP aggregation is determined by different types of interactions and represents an intrinsic property of the protein that appears to be necessary for its functioning in vivo.     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Background

Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure.

Results

Transfection conditions were first optimized to achieve expression levels between 10 and 18?mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4–9?mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (K_d) of 5?nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured k_cat (7.2?±?0.5?min^??1) and K_M (1.2?±?0.3?μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption.

Conclusions

The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher k_cat and K_M values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

     

Cloning of the <Emphasis Type="Italic">Penicillium canescens</Emphasis> endo-1,4-β-glucanase gene <Emphasis Type="Italic">egl3</Emphasis> and the characterization of the recombinant enzyme

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-β-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for β-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54°C, respectively. The K _m and V _m values for CMC hydrolysis were determined; they amounted to 17.1 g/l and 0.31 μM/(mg s), respectively.     

Cloning and expression of <Emphasis Type="Italic">β</Emphasis>-glucosidase gene from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> into <Emphasis Type="Italic">E. coli</Emphasis> BL 21 (DE3)

A 1.4 Kb fragment of Bacillus licheniformis ATCC 14580 encoding β-glucosidase was cloned and expressed in Escherichia coli. β-Glucosidase expressed by E. coli harboring cloned gene was located entirely in the intracellular fraction. Recombinant β-glucosidase protein was purified to homogeneity level and the molecular weight was found to be 53 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It gave maximum activity at 50°C and pH 6. K _m and V _max were 0.206 mM and 1.26 U/mg, respectively, with p-nitrophenyl-β-D-glucopyranoside, while activation energy E_a, enthalpy of activation ?H and entropy of activation ΔS were found to be 66.31 kJ/mol, 64.04 kJ/mol and 48.28 J/mol/K, respectively. The pK_a1 and pK_a2 of the ionizable groups of active site residues involved in V_max were found to be 5.5 and 7.0, respectively. When the recombinant β-glucosidase protein was used as a member of consortium with endoglucanase and exoglucanase for the saccharification of wheat straw, 123% increase in saccharification was observed.     

Synthesis of Bacteriophage λ Proteins <Emphasis Type="Italic">in vitro</Emphasis>

λ DNA stimulates a low level of protein synthesis in extracts of E. coli supplemented with RNA polymerase. The synthesis is efficiently and specifically repressed by addition of purified λ repressor. About 90% of the protein product is from the rightwards, or tof, promoter (P _R ) and 10% from the leftwards, or N, promoter (P _L ). The main polypeptide product is a low molecular species from the tof operon.     

Purification and characterization of a β-1,4-glucosidase from a newly isolated strain of <Emphasis Type="Italic">Fomitopsis pinicola</Emphasis>

An efficient ß-1,4-glucosidase (BGL) producing strain, Fomitopsis pinicola KMJ812, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular BGL was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The relative molecular weight of F. pinicola BGL was determined to be 105 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, or 110 kDa by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 4.5 and a temperature optimum of 50°C. The enzyme showed high substrate specificity and high catalytic efficiency (k _cat?=?2,990 s^?1, K _m?=?1.76 mM, k _cat/K _m?=?1,700 mM^?1 s^?1) for p-nitrophenyl-β-d-glucopyranoside. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 3, indicating that the F. pinicola BGL is a member of glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, F. pinicola BGL is distinguished from other BGLs by its high catalytic efficiency and strict substrate specificity.     

Highly immunogenic variant of attenuated vaccinia virus

The LIVPΔ6 strain of vaccinia virus (VACV) was created by genetic engineering on the basis of previously obtained attenuated 1421ABJCN strain by target deletion of the A35R gene encoding an inhibitor of antigen presentation by the major histocompatibility complex class II. 1421ABJCN is the LIVP strain of VACV with five inactivated virulence genes encoding hemagglutinin (A56R), γ-interferon-binding protein (B8R), thymidine kinase (J2R), complement-binding protein (C3L), and Bcl2-like inhibitor of apoptosis (N1L). The highly immunogenic LIVPΔ6 strain could be an efficient fourth-generation attenuated vaccine against smallpox and other orthopoxvirus infections.     

Enzymological properties of endo-(1–4)-β-glucanase Eg12p of <Emphasis Type="Italic">Penicillium canescens</Emphasis> and characteristics of structural gene <Emphasis Type="Italic">egl2</Emphasis>

Gene egl2 of secreted endo-(1–4)-β-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding β-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60°C, respectively, exhibited specific activity of 33 IU, and had K _m and V _max in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 μmol/sec per mg, respectively.     

Molecular characteristics and extracellular expression analysis of farnesyl pyrophosphate synthetase gene in <Emphasis Type="Italic">Inonotus obliquus</Emphasis>

A farnesyl pyrophosphate synthase gene was cloned from Inonotus obliquus, designated IOFPS. The IOFPS cDNA contained an open reading frame (ORF) of 972 bps, encoding a protein of 324 amino acids. The deduced amino acid sequence of IOFPS revealed moderate homology with that of other fungi, and contained four conserved domains. Phylogenetic analysis showed that IOFPS belonged to the basidiomycete group. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the IOFPS gene was successfully expressed in a yeast recombinant cell. Enzyme catalytic experiments were carried out with purified protein (IOFPS protein), which was isolated and purified from recombinant yeast cells. The special hydrolysis product (farnesol) was then detected by liquid chromatography coupled with tandem mass spectrometry (LC-MS). These results indicated that the cloned cDNA encoded a farnesyl diphosphate synthase and the IOFPS protein maintained catalytic activity in vitro.     

Tissue specific expression of hepatitis B virus surface antigen in transgenic plant cells and tissue culture

The tobacco plants (Nicotiana tabacum L.) carrying the HBsAg gene controlled by (Aocs)₃AmasPmas, the hybrid promoter that includes regulatory elements of the agrobacterial octopine and mannopine synthase genes, as well as plants controlled by the same promoter and adh1, maize alcohol dehydrogenase gene intron were obtained. The presence of the adh1 gene intron did not significantly change the level of expression of the HBsAg gene in plants. The analysis of expression of hepatitis B virus surface antigen (HBs-antigen) in transformed plants expressing the HBsAg under the control of different promoters was made. The level of HBs-antigen in plants carrying the HBsAg gene controlled by (Aocs)₃AmasPmas, the hybrid agrobacterium-derived promoter, was the highest in roots and made up to 0.01% of total amount of soluble protein. The level of HBs-antigen in plants carrying the HBsAg gene controlled by the dual 35S RNA cauliflower mosaic virus promoter was the same in all organs of the plant and made up to 0.06% of the total amount of soluble protein. Hairy root and callus cultures of plants carrying the HBsAg gene and expressing the HBs-antigen were obtained.     

High-yield expression,purification, characterization,and structure determination of tag-free <Emphasis Type="Italic">Candida utilis</Emphasis> uricase

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2₁2₁2₁ with unit cell parameters a?=?69.16 Å, b?=?139.31 Å, c?=?256.33 Å, and α?=?β?=?γ?=?90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.     

Subtilosin Prevents Biofilm Formation by Inhibiting Bacterial Quorum Sensing

Subtilosin, the cyclic lantibiotic protein produced by Bacillus subtilis KATMIRA1933, targets the surface receptor and electrostatically binds to the bacterial cell membrane. In this study, subtilosin was purified using ammonium sulfate ((NH₄)₂SO₄) precipitation and purified via column chromatography. Subtilosin’s antibacterial minimum and sub-minimum inhibitory concentrations (MIC and sub-MIC) and anti-biofilm activity (biofilm prevention) were established. Subtilosin was evaluated as a quorum sensing (QS) inhibitor in Gram-positive bacteria using Fe(III) reduction assay. In Gram-negative bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter. The results showed that Gardnerella vaginalis was more sensitive to subtilosin with MIC of 6.25 μg/mL when compared to Listeria monocytogenes (125 μg/mL). The lowest concentration of subtilosin, at which more than 90% of G. vaginalis biofilm was inhibited without effecting the growth of planktonic cells, was 0.78 μg/mL. About 80% of L. monocytogenes and more than 60% of Escherichia coli biofilm was inhibited when 15.1 μg/mL of subtilosin was applied. Subtilosin with 7.8–125 μg/mL showed a significant reduction in violacein production without any inhibitory effect on the growth of C. violaceum. Subtilosin at 3 and 4 μg/mL reduced the level of Autoinducer-2 (AI-2) production in G. vaginalis. However, subtilosin did not influence AI-2 production by L. monocytogenes at sub-MICs of 0.95–15.1 μg/mL. To our knowledge, this is the first report exploring the relationship between biofilm prevention and quorum sensing inhibition in G. vaginalis using subtilosin as a quorum sensing inhibitor.     

Production of proinsulin in marker-free transgenic tobacco plants using <Emphasis Type="Italic">CRE</Emphasis>/loxP system

The demand for INSULIN is increasing rapidly along with the increased number of diabetic patients. Using the CRE/loxP system, we developed a selective marker-free system without crossing to produce PROINSULIN in transgenic plant. In frame of this approach, the induced promoter pRD29A was isolated from Arabidopsis. The CRE recombinase gene was placed under the control of pRD29A between two loxP recombination sites together with the selective NPTII gene. Furthermore, the binary vector with CRE recombinase and PROINSULIN was constructed and introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. Gene excision was used to remove the sequence between the two loxP sites at the presence of 200 mM NaCl. PCR analysis showed that self-excision occurred in several T₀ transgenic plants. Transgenic plants without any marker gene successfully expressed PROINSULIN. This auto-excision strategy provides efficient means of removing the selectable marker gene from transgenic plants. It is an efficient method for producing bio-safe recombinant protein and other valuable substances in plants.     

Effect of influenza A virus and bacterial lipopolysaccharide on proliferation and expression of cytokines and other cellular factors in the endothelial cell line ECV-304

Viral infection and bacterial lipopolysaccharide (LPS) cause endothelial-cell dysfunction. The aim of the current study was to investigate the effect of influenza A virus and LPS from Escherichia coli on the proliferative activity and gene expression of cytokines and cellular factors (TNFα, TGFβ, IFN-γ, MMP-9, NF-κB, Rho A, eNOS, and iNOS) in human endothelial cells ECV-304. It was found that ECV-304 cells infected with very low infectious doses of influenza virus acquired the capacity for the long-term active proliferation (over eight passages). Addition of LPS from E. coli reduced the virus-stimulated cell proliferation. It was shown that influenza virus and LPS affected the gene expression of cytokine and other cellular factors. When endothelial cells were infected with influenza A virus in the presence of LPS, there was a significant increase in the expression of several genes and the expression pattern of certain genes was modified. Expression of MMP-9 gene inhibited by the virus and LPS separate exposure significantly increased during the first day after addition of the virus and LPS simultaneously. The same was true for the IFN-γ gene expression. TNFα gene was active only for 1–3 days whereas the expression of TGFβ, eNOS, iNOS, NF-κB and Rho A genes increased significantly on the fifth day, as it was observed with the cells treated with LPS only. Thus, the influenza A virus and LPS change the physiological state of endothelial cells. This occurred during various time periods (as well as at various degrees of viral infection) produced by different cellular factors and, possibly, involved different signaling pathways.     

Construction,Expression, and Characterization of Recombinant Pfu DNA Polymerase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His₆-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni²⁺ chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.