Isolation and Purification of Recombinant HBsAg of Human Hepatitis B Virus from Silkworm Larvae

Recombinant HBsAg coded by preS1-preS2-S regions of hepatitis B virus was expressed in Bombyx mori silkworm larvae. Recombinant HBsAg was expressed (30–40 μg/mL, 0.1% of the total amount of extracted protein) by larvae infected with recombinant baculovirus rBmNPV-Hep-preS1-S containing cDNA of HBsAg strictly by the polyhedrin gene promoter. Recombinant HBsAg consisting of a polypeptide of molecular weight ∼36 kDa (p36) was purified by gel filtration and affinity chromatography to 92% purity. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 477–480, September–October, 2005.     

Immunochemical Study of Antigenic Determinants of Recombinant HBsAg Produced by Bombyx mori Larvae

Antibodies to various protein fractions isolated from larvae of mulberry silkworm containing recombinant HBsAg, to plasma HBV, and to healthy human serum were obtained. The cross reactivity of the antibodies was studied using enzyme-linked immunosorbent assay (ELISA). The antigenic determinants of recombinant HBsAg are identical to those in plasma and have practically no common antigenic determinants with healthy human blood serum     

Production and Characterization of Polyclonal and Monoclonal Abs Against the RNA-Binding Protein QKI

RNA-binding protein QKI, a member of the Signal Transduction and Activation of RNA family, is found to be essential in the blood vessel development and postnatal myelination in central nervous system (Woo et al., Oncogene 28:1176–1186, 2009; Lu et al., Nucleic Acids Res 31(15):4616–4624, 2003; Bohnsack et al., Genesis 44(2):93–104, 2006). However, its wide expression pattern suggests other fundamental roles in vivo (Kondo et al., Mamm Genome 10(7):662–669, 1999). To facilitate the understanding of QKI function in various systems, we prepared the polyclonal and monoclonal antibodies against QKI. To obtain the antigen, recombinant His-tagged QKI was expressed in Escherichia coli and highly purified by Ni²⁺-chelated column combined with hydrophobic and ion exchange methods. Following three types of immunizations with different adjuvants, including Freund’s, PAGE gel, and nitrocellulose membrane, only the antiserum produced with Freund’s adjuvant is effective for Western blot detection. Several McAb clones are able to recognize both endogenous and over-expressed QKI with high affinity in Western blot and immunofluorescence. The specificity of Ab was validated as weakening, and no specific signals were observed in cells with QKI knocking down. Immunohistochemistry analysis further showed positive staining of QKI in kidney where QKI mRNA was abundantly expressed, ensuring the wide applications of the QKI Abs in the ongoing mechanistic studies.     

Expression,Purification, and Characterization of Hepatitis B Virus Surface Antigens (HBsAg) in Yeast <Emphasis Type="Italic">Pichia Pastoris</Emphasis>

Prevention of the prevalence of HB depends upon the development of efficient diagnostic reagent and preventive vaccine. Pichia pastoris offers many advantages over the other expression systems in the production of recombinant HBsAg. In this study, we reported that the recombinant P. pastoris strains were cultured in shake flasks and then scaled up in a 5.0-l bioreactor: approximately 27 mg/l of the protein and the maximal cell OD at 600 nm of 310 were achieved in the bioreactor. The recombinant HBsAg was purified by three steps of purification procedures. SDS-PAGE showed that the purified recombinant HBsAg constituted only one homogeneous band of ~24 kDa. CsCl density gradient ultracentrifugation assay indicated that the density of the HBsAg was 1.2 mg/ml, which was in agreement with the natural HBsAg, the HBsAg expressed in Saccharomyces cerevisiae and in mammalian cells. Electron microscope observation revealed that the purified recombinant HBsAg was homogeneous 22-nm particles, suggesting the HBsAg expressed in P. pastoris was self-assembled to virus-like structures. Competitive ELISA indicated that P. pastoris-derived HBsAg possessed the excellent immunoreaction with anti-HBsAg. Animal immunization showed that the immunogenicity of P. pastoris-derived HBsAg was superior to that of S. cerevisiae-derived HBsAg. Together, our results demonstrated that the recombinant HBsAg expressed in P. pastoris could provide promising, inexpensive, and large-scale materials for the diagnostic reagent and vaccine to prevent HBV infection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Rushi Liu and Qinlu Lin contributed equally to this work.     

Expression,Purification, and Antibacterial Activity of Bovine Lactoferrampin–Lactoferricin in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N₁ domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA–LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA–LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA–LFA–LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA–LFC peptide was purified via two-step column chromatography and identified by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA–LFC. The fermentation supernate and the purified LFA–LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA–LFC in P. pastoris and may have potential practical applications in microbial peptide production.     

Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision–mass spectrometry

The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision–mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrP^C) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision–MS, with complete suppression of nonspecific bPrP binding.     

Europium-Containing Organic Gels and Organic and Carbon Aerogels. Preparation and Initial Applications in Catalysis

Summary. We prepared organic gels and organic and carbon aerogels doped with europium through sol–gel processes. Eu-gels were prepared by sol–gel polymerization of the potassium salt of 2,4-dihydroxybenzoic acid with formaldehyde followed by ion-exchange with Eu(OTf)₃. Eu–organic aerogels were obtained after CO₂ supercritical drying of the gels and Eu–carbon aerogels were obtained by pyrolysing the organic aerogels. The Eu-gels containing 12% europium proved to be efficient as recoverable catalyst in Michael additions. Deceased on February 20, 2006     

Europium-Containing Organic Gels and Organic and Carbon Aerogels. Preparation and Initial Applications in Catalysis

We prepared organic gels and organic and carbon aerogels doped with europium through sol–gel processes. Eu-gels were prepared by sol–gel polymerization of the potassium salt of 2,4-dihydroxybenzoic acid with formaldehyde followed by ion-exchange with Eu(OTf)₃. Eu–organic aerogels were obtained after CO₂ supercritical drying of the gels and Eu–carbon aerogels were obtained by pyrolysing the organic aerogels. The Eu-gels containing 12% europium proved to be efficient as recoverable catalyst in Michael additions.     

Recombinant firefly luciferase in Escherichia coli

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.     

Highly sensitive potentiometric immunosensor for hepatitis B surface antigen diagnosis

Immunosenors are of great interest because oftheir potential utility as specific, simple, label-free anddirect detection techniques and reductions in size, costand time of analysis compared with conventional im-munoassay techniques. The immunoassays with …     

Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR

Vitellogenin (VTG) and choriogenin (CHO) are valuable biomarkers of endocrine-disrupting compound (EDC) exposure in fish. Existing immunoassays are limited to a few species, which restricts their use for the analysis of local wildlife sentinels. Using C. facetum as a relevant South American model fish, this work presents a new strategy for the preparation of antibodies to VTG and CHO, with zero cross-reactivity with fish serum components. Recombinant fragments of Cichlasoma facetum VTG (280-mer) and CHO (223-mer) were prepared by degenerate primer RT-PCR and expression in E. coli. Polyclonal and monoclonal antibodies prepared with these antigens were used to develop rapid dotblot assays for VTG and CHO. Both the polyclonal and monoclonal antibodies prepared with the recombinant antigens reacted against the native proteins adsorbed on to nitrocellulose allowing the set up of sensitive dotblot assays. The VTG assay was further validated with spiked samples and purified native VTG. Exposure experiments with several estrogenic compounds revealed the potential of C. facetum as a sensitive biomonitor that produced measurable responses at concentrations of 100 ng L⁻¹ of 17-beta-estradiol, 100 ng L⁻¹ of ethynylestradiol, and 6.6 μg L⁻¹ of nonylphenol. The approach described here may be applied to other native species to produce highly specific and sensitive rapid tests. It may be particularly advantageous for species that cannot be kept in captivity or when homogeneous purification of the immunizing proteins is particularly challenging. In conclusion, we present a novel approach to develop a strategy for the generation of immunoassay reagents for vitellogenin (VTG) and choriogenin (CHO), which will facilitate regional studies on the impact of endocrine-disrupting chemicals on local wildlife.     

Thermal stability of recombinant green fluorescent protein (GFPuv) at various pH values

The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5–9.0 μg of GFPuv/mL) was exposed to various pH conditions (4.91–9.03) and temperatures (75–95°C) in the 10 mM buffers: acetate (pH 5.0–7.0), phosphate (pH 5.5–8.0), and Tris-HCl (pH 7.0–9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85°C, the D-values (pH 7.1–7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate) The stability of GFPuv in Tris-HCl (pH>8.0) was constant at 90 and 95°C, and the D-values were 7.93 (pH 8.38–8.92) and 6.0 min (pH 8.05–8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100°C.     

Polyfunctional ion-exchangers based on wood

α-Oxide wood derivatives were synthesized by O-alkylation of wood with a bifunctional epoxide oligomer in the presence of Et₃N catalyst. Polyfunctional ion-exchangers were prepared by subsequent amination of them by aliphatic polyamines. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 480–483, September–October, 2006.     

Analysis of erythritol in foods by polyclonal antibody-based indirect competitive ELISA

Sugar alcohols are widely used as food additives and drug excipients. Erythritol (INS 968) is an important four-carbon sugar alcohol in the food industry. Erythritol occurs naturally in certain fruits, vegetables, and fermented foods. Currently, HPLC and GC methods are in use for the quantification of erythritol in natural/processed foods. However, an immunoassay for erythritol has not been developed so far. We have utilized affinity-purified erythritol-specific antibodies generated earlier [9] to develop an indirect competitive ELISA. With erythritol–BSA conjugate (54 mol/mol; 100 ng/well) as the coating antigen, a calibration curve was prepared using known amounts of standard meso-erythritol (0.1–100,000 ng) in the immunoassay. Watermelon (Citrullus lanatus) and red wine were selected as the food sources containing meso-erythritol. The amount of meso-erythritol was calculated as 2.36 mg/100 g fresh weight of watermelon and 206.7 mg/L of red wine. The results obtained from the immunoassay are in close agreement with the reported values analyzed by HPLC and GC (22–24 mg/kg in watermelon and 130–300 mg/L in red wine). The recovery analyses showed that added amounts of meso-erythritol were recovered fairly accurately with recoveries of 86–105% (watermelon) and 85–93.3% (red wine). The method described here for erythritol is the first report of an immunoassay for a sugar alcohol. Figure Indirect competitive ELISA for quantitation of erythritol     

Catalytic antibodies as a new generation of biocatalysts

The present state and perspectives of the creation of a new generation of biocatalysts, catalytic antibodies (CA), and their application to organic synthesis are discussed. The problems to be solved for the development of the practical application of CA are especially noted. This paper was prepared in response to a request from the Editorial board in order to attract the attention of investigators to the creation of artificial enzymes, called catalytic antibodies, which is a new and important direction in science. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1568–1574, September, 1997     

Syntheses and properties of poly (amide–imide)s based on 5,5′-bis[4-(4-trimellitimidophenoxy)phenyl]-hexahydro-4,7-methanoindan and aromatic diamines

 A novel polymer-forming diimide–diacid, 5,5′-bis[4-(4-trimellitimido phenoxy)phenyl]-hexahydro-4,7-methanoindan (II), was prepared by the condensation reaction of 5,5′-bis[4-(4-aminophenoxy)phenyl]-hexahydro-4,7-methanoindan with trimellitic anhydride. A series of novel aromatic poly(amide–imide)s (PAIs) containing polycyclic cardo groups was prepared by the direct polycondensation of II with various aromatic diamines using phosphorylation techniques. The polymers had inherent viscosities between 0.71 and 0.96 dl/g. The polymers were soluble in polar solvents such as N-methyl-2-pyrrolidone, N,N-dimethylacetamide (DMAc) and N,N-dimethylformamide, and could be cast from their DMAc solutions into transparent, flexible, and tough films, except for III _a . These films had yield strengths of 85–114 MPa, tensile strengths of 77–102 MPa, an elongation at break of 8–17%, and initial moduli of 2.0–2.7 GPa. Wide-angle X-ray diffraction revealed that the polymers are amorphous. The glass-transition temperatures of the polymers were in the range 242–312 °C. All the PAIs exhibited no appreciable decomposition below 430 °C, and their 10%-weight-loss temperatures were in the range 484–507 °C in nitrogen and 494–515 °C in air. Received: 26 January 1999 Accepted in revised form: 11 May 1999     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-methyl urocanates of hydroxyderivatives of isocembrol

Alcohols were prepared by stereospecific hydroxylation of isocembrol and were esterified into N-methylurocanates, proposed biomimetics of taxol. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 119–123, March–April, 2007.     

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.     

Structural and thermal properties of monolithic silica–phosphate (SiO<Subscript>2</Subscript>–P<Subscript>2</Subscript>O<Subscript>5</Subscript>) gel glasses prepared by sol–gel technique

A sol–gel process for producing monolithic silica–phosphate (SiO₂–P₂O₅) system different concentrations of P₂O₅, starting with tetra-ethoxysilane TEOS, and triethyl-phosphate as sources of SiO₂ and P₂O₅ was performed. The gels were heat-treated at temperatures ranging from 100 up to 900 °C. The structural and chemical analyses of the samples were determined by using X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR). It was found from the XRD that the existence of phosphorus enhances the crystallization of silica gel, while the FTIR indicated the main functional groups of silica–phosphate. It is important to study the effect of hydroxyl in silica–phosphate glass. The results obtained are promising to use the prepared samples in a variety of applications, ranging from traditional application such as lighting products) to the modern application (such as optical fibers. Optical studies were measured by using the spectrophotometer in wavelength range 0.2–2.5 μm. The refractive index (n) was calculated for the prepared samples, it was found to be strongly affected by structural rearrangement resulting from the elimination of the solvent and the Si–OH, Si–O–Si and Si–O–OH bonding by phosphate and aluminum and it increases by increasing phosphate concentrations. The weight losses have investigated for prepared samples.     

Solid-Phase Synthesis of a Biotin Derivative and its Application to the Development of Anti-Biotin Antibodies

A biotin derivative, namely biotin–aminocaproic acid–lysine (BAL), was synthesized with solid-phase chemistry, conjugated to a carrier-protein, and used for rabbit immunization. The aminocaproic acid–lysine “long-arm” was used in order to project the biotin-hapten above the carrier-protein surface. Lysine was selected due to its N^ε-amino group, through which BAL was conjugated to the carrier-protein. BAL was synthesized on a commercially available resin with the Fmoc-solid-phase strategy; this has simplified the experimental procedure, overcome the need for intermediate purification steps, and led to a final product of high purity, with high yield. The anti-BAL antibodies recognized free biotin, as shown with an in-house-developed ELISA, in which biotin conjugated to a synthetic “lysine–dendrimer” was used to coat the ELISA microwells. In immunocytology and Western-blot experiments, the anti-BAL antibodies led to similar results with those obtained with streptavidin. Synthetic derivatives of hapten molecules that can be easily prepared with solid-phase chemistry, such as BAL, may be used for the development of specific antibodies for the corresponding hapten.