Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in <Emphasis Type="Italic">Bombyx mori</Emphasis> Chrysalides

Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.     

Cloning of Thermostable Cellulase Genes of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> and Their Secretive Expression in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.     

High-Level Expression and Efficient Purification of Bioactive Swollenin in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The bioactivity of swollenin is beneficial to cellulose decomposition by cellulase despite the lack of hydrolytic activity itself. In order to improve the productivity of swollenin, the effects of culture conditions on the expression level in recombinant Aspergillus oryzae were investigated systematically. With regard to the bioactivity of swollenin, glycerin and peanut meal were the optimal carbon or nitrogen source, respectively. The highest level production of swollenin (50 mg L⁻¹) was attained after 88 h cultivation with the initial pH of 5.6 in the culture medium. Then the soluble swollenin was effectively purified from the cultural supernatant by ammonium sulfate precipitation and cationic exchange chromatography with recovery yield of 53.2%. The purified swollenin was fully bioactive due to its strong synergistic activity with cellulose.     

Functional Expression of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Protective Antigen in <Emphasis Type="Italic">E. coli</Emphasis>

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.     

Molecular Cloning and Expression Analysis of an <Emphasis Type="Italic">Mn</Emphasis>-<Emphasis Type="Italic">SOD</Emphasis> Gene from <Emphasis Type="Italic">Nelumbo nucifera</Emphasis>

A rapid amplification cDNA end (RACE) assay was established to achieve the complete sequence of mitochondrial manganese-superoxide dismutase (Mn-SOD) cDNA in Nelumbo nucifera. The obtained full-length cDNA of Mn-SOD was 926 bp and contained a 699-bp open reading frame encoding an Mn-SOD precursor of 233 amino acids. The recombinant of Mn-SOD expressed by PET-32a vector in Escherichia coli BL21 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting assays. A 3D structural model of the Mn-SOD was constructed by homology modeling. Real-time polymerase chain reaction analysis revealed that Mn-SOD mRNA was expressed in young leaves, blossom, stems, and terminal buds during reproductive stage but with the highest expression in young leaves. This significant difference demonstrated the differential expression of Mn-SOD in various organs of N. nucifera.     

Expression of a <Emphasis Type="Italic">hemA</Emphasis> Gene from <Emphasis Type="Italic">Agrobacterium radiobacter</Emphasis> in a Rare Codon Optimizing <Emphasis Type="Italic">Escherichia coli</Emphasis> for Improving 5-aminolevulinate Production

The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.     

Cloning,Expression, and Characterization of a Novel (<Emphasis Type="Italic">S</Emphasis>)-Specific Alcohol Dehydrogenase from <Emphasis Type="Italic">Lactobacillus kefir</Emphasis>

A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-β-d-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.     

Biocatalytic Properties of a Recombinant <Emphasis Type="Italic">Fusarium proliferatum</Emphasis> Lactonase with Significantly Enhanced Production by Optimal Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The levo-lactonase gene of Fusarium proliferatum ECU2002 (EC3.1.1.25) was cloned and expressed in Escherichia coli JM109 (DE3) for biocatalytic resolution of industrially important chiral lactones, including DL-pantoyl lactone which was a key precursor to calcium d-pantothenate. By increasing the biomass concentration and lowering the inducer (isopropyl-β-d-thiogalactoside) concentration and induction temperature, the lactonase production was significantly enhanced up to 20 kU/L, which was 20 times higher than that of wild-type strain F. proliferatum ECU2002. The recombinant Fusarium lactonase was purified using immobilized metal affinity chromatography, and its SDS-PAGE revealed a molecular mass of 50 kDa for the recombinant protein, suggesting that the enzyme was a simplex protein. Furthermore, biocatalytic properties of the recombinant lactonase were investigated, including kinetic parameters, additive’s effect, and substrate specificity. The results reported in this paper provide a feasible method to make the whole cells of E. coli JM109 (DE3) expressing lactonase gene to be a highly efficient and easy-to-make biocatalyst for asymmetric synthesis of chiral compounds.     

Properties and <Emphasis Type="Italic">N</Emphasis>-glycosylation of recombinant endoglucanase II from <Emphasis Type="Italic">Penicillium verruculosum</Emphasis>

Cellulases are the main components of enzyme complexes used in biotransformation processes of plant raw materials into valuable commercial products. Endoglucanase II (EG II) from the Penicillium verruculosum fungus was cloned into Penicillium canescens. The homogeneous recombinant EGII form is isolated and its properties are studied in comparison with the native enzyme. The N-glycosylation sites and the structure of the N-linked glycans are been determined using mass spectrometry. The biochemical and catalytic properties, as well as the N-glycosylation type of the obtained recombinant EGII form, appear to be close to the native enzyme. At the two potential N-glycosylation sites (N42 and N194) of both forms of the enzyme, N-linked high mannose glycans (or their enzymatic “trimming” products) according to the general formula (Man)_1–9(GlcNAc)₂ are detected. No glycosylation is found at the third potential site (N19).     

Molecular properties of the copolymers of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride and maleic acid

The hydrodynamic and conformational properties of molecules of poly(N,N-diallyl-N,N-dimethylammonium chloride) and N,N-diallyl-N,N-dimethylammonium chloride-maleic acid copolymers of different compositions in solutions with various ionic-strength and pH values, as well as of the polyelectrolyte complex based on the copolymer with dodecyl sulfate anions in chloroform, are studied. For poly(N,N-diallyl-N,N-dimethylammonium chloride) molecules in a 1 M NaCl solution, the Kuhn segment length and the hydrodynamic diameter of the chain are estimated as A = 3.9 nm and d = 0.48 nm, respectively. In acidic solutions with pH 3.5, the copolymers demonstrate behavior typical for polyelectrolytes. In an alkaline solution with pH 13, when 1 M NaCl is added to the solution of the copolymer containing 29 mol % maleic acid units, there is an antipolyelectrolyte effect that manifests itself as an increase in the intrinsic viscosity of the copolymer and in the hydrodynamic radius of its molecules. It is found that an increase in the fraction of maleic acid units in the copolymer from 12 to 42 mol % brings about a reduction in the equilibrium rigidity of its macromolecules from 4.1 to 2.2 nm. The equilibrium rigidity of polyelectrolyte-complex molecules is higher than that of initial copolymer molecules owing to steric interactions arising between the aliphatic chains of dodecyl sulfate anions. In an electric field, the molecules of the complex are oriented owing to the induced dipole moment resulting from the displacement of dodecyl sulfate anions along the chain contour.     

Synthesis of epothilones molecule fragment (15<Emphasis Type="Italic">R</Emphasis>)-C<Superscript><Emphasis Type="Italic">13</Emphasis></Superscript>-C<Superscript><Emphasis Type="Italic">21</Emphasis></Superscript> from D-mannitol

Efficient synthesis of an epothilone molecules fragment (15R)-C¹³-C²¹ was carried out from D-mannitol through its conversion into methyl 2,3-O-cyclohexylidene-D-glycerate followed by the cyclopropanation of the ester group with ethylmagnesium bromide in the presence of titanium(IV) isopropoxide and the transformation of the obtained cyclopropanol into the corresponding 2-substituted allyl bromide. The coupling of the latter catalyzed by copper with 2-methylthiazolyl-4-magnesium bromide, the shift of a double carbon-carbon bond in the product obtained into the position, conjugated with the thiazole ring, and the common transformation of the protected 1,2-diol function afforded the target compound in 15% yield.     

Extracellular Production and Characterization of Two <Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Asparaginases

l-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four l-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Tandem Multimer Expression and Preparation of Hypoglycemic Peptide MC6 from <Emphasis Type="Italic">Momordica charantia</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Tandem repeat multimers of Momordica charantia (MC) peptide MC6 were designed and the recombinant plasmid containing 10 copies of MC6 gene was constructed to improve the expression level of MC6 in Escherichia coli. Under the selected conditions of cultivation and induction, the expression level of recombinant TrxA–MC6₁₀ protein was above 25% of total bacteria protein. This fusion protein was purified and cleaved with HCl (13%, w/v). Either the un-cleaved or cleaved recombinant proteins was analyzed pharmacological activity by alloxan-induced diabetic mice and only the cleaved products of the recombinant protein showed significant hypoglycemic effects. The study provides a convenient and economical method for the large-scale production of anti-diabetic medicines for pharmaceutical applications.     

Engineering <Emphasis Type="Italic">Pichia pastoris</Emphasis> for Efficient Production of a Novel Bifunctional <Emphasis Type="Italic">Strongylocentrotus purpuratus</Emphasis> Invertebrate-Type Lysozyme

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960 U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5 mg/L and purity of >?99%. Recombinant sp-iLys reached its peak lytic activity of 8560 U/mg at pH 6.0 and 30 °C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 °C with the presence of 0.05 M Na⁺. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.     

Cloning,Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. K in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg⁻¹ at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K _m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced by Na⁺, NH₄⁺, Mg²⁺, Fe²⁺, Fe³⁺, Co²⁺, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents.     

Redox Behavior of Neptunium(V) in Tributyl Phosphate and <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-Dihexyl Octanamide Extractants Dissolved in <Emphasis Type="Italic">n</Emphasis>-Dodecane

Spectrophotometric investigations have been carried out on the disproportionation of Np(V) to form Np(IV) and Np(VI) in 1.1 mol⋅L⁻¹ solutions of tributyl phosphate (TBP) and in N,N-dihexyl octanamide (DHOA) in n-dodecane medium. The Np(V) was found to coordinate with Np(IV) in 1.1 mol⋅L⁻¹ TBP solution in n-dodecane to form a mixed valence “cation–cation” complex by bonding through an axial oxo group on Np(V). By contrast, this interaction was less prominent in the case of 1.1 mol⋅L⁻¹ DHOA solutions. The effect of 1-octanol, added as phase modifier, on the disproportionation behavior of Np(V) was also investigated. An attempt was made to calculate the disproportionation/reduction rate constants for Np(V) under the conditions of these studies. Absorbance measurements on the Np stripped from organic phases revealed the occurrence of Np(V) in the aqueous phase.     

Enhanced Production of <Emphasis Type="SmallCaps">l</Emphasis>-Arginine by Expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> Hemoglobin Using a Novel Expression System in <Emphasis Type="Italic">Corynebacterium crenatum</Emphasis>

Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g⁻¹, and the oxygen uptake rates reached 0.25 mg A₅₆₂⁻¹ h⁻¹ which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L⁻¹, and the biomass was largely increased to 6.45 g L⁻¹, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products.