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PubMed2004

Efficient production of recombinant human transcription factor IIE.

Transcription factor IIE (TFIIE) is a general initiation and promoter escape factor for RNA polymerase II composed of p56 (TFIIE-alpha) and p34 (TFIIE-beta) subunits. Our laboratories experienced difficulty producing adequate quantities of recombinant human TFIIE-alpha for in vitro studies using available clones. We therefore re-engineered the TFIIE subunit production vectors and tested various Escherichia coli host strains to optimize expression. We report a much-improved system for production of pure, soluble, and active TFIIE complex for in vitro studies.

Cloning, Molecular↗
PubMed2002

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine.

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.

Administration, Oral↗
PubMed2000

Sequence and expression of a halobacterial beta-galactosidase gene.

Studies of gene expression in haloarchaea have been greatly hindered by the lack of a convenient reporter gene. In a previous study, a beta-galactosidase from Haloferax alicantei was purified and several peptide sequences determined. The peptide sequences have now been used to clone the entire beta-galactosidase gene (designated bgaH) along with some flanking chromosomal DNA. The deduced amino acid sequence of BgaH was 665 amino acids (74 kDa) and showed greatest amino acid similarity to members of glycosyl hydrolase family 42 [classification of Henrissat, B., and Bairoch, A. (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293: 781-788]. Within this family, BgaH was most similar (42-43% aa identity) to enzymes from extremely thermophilic bacteria such as Thermotoga and Thermus. Family 42 enzymes are only distantly related to the Sulfolobus LacS and Escherichia coli LacZ enzymes (families one and two respectively). Three open reading frames (ORFs) upstream of bgaH were readily identified by database searches as glucose-fructose oxidoreductase, 2-dehydro-3-deoxyphosphogluconate aldolase and 2-keto-3-deoxygluconate kinase, enzymes that are also involved in carbohydrate metabolism. Downstream of bgaH there was an ORF which contained a putative fibronectin III motif. The bgaH gene was engineered into a halobacterial plasmid vector and introduced into Haloferax volcanii, a widely used strain that lacks detectable beta-galactosidase activity. Transformants were shown to express the enzyme; colonies turned blue when sprayed with Xgal and enzyme activity could be easily quantitated using a standard ONPG assay. In an accompanying publication, Patenge et al. (2000) have demonstrated the utility of bgaH as a promoter reporter in Halobacterium salinarum.

Amino Acid Sequence↗
PubMed1999

Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecular cloning and functional expression of stachyose synthase.

Stachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 2.4.1.67). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT-PCR, followed by rapid amplification of cDNA ends (RACE-PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.

Amino Acid Sequence↗
PubMed1999

Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves.

To evaluate the expression of different forms of a tumor-specific antibody in plants, we adapted a recently described Agrobacterium-mediated transient expression system. A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse/human chimeric antibody (cT84.66) derived from the parental murine mAb T84. 66 specific for the human carcinoembryonic antigen were engineered into a plant expression vector. Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors. In vivo assembly of full-size cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week. His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography. Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS/PAGE, Western blotting, and ELISA.

Animals↗
PubMed1998

Protection against respiratory syncytial virus infection by DNA immunization.

Respiratory syncytial virus (RSV) remains a major cause of morbidity and mortality in infants and the elderly and is a continuing challenge for vaccine development. A murine T helper cell (Th) type 2 response associates with enhanced lung pathology, which has been observed in past infant trials using formalin-inactivated RSV vaccine. In this study, we have engineered an optimized plasmid DNA vector expressing the RSV fusion (F) protein (DNA-F). DNA-F was as effective as live RSV in mice at inducing neutralizing antibody and cytotoxic T lymphocyte responses, protection against infection, and high mRNA expression of lung interferon gamma after viral challenge. Furthermore, a DNA-F boost could switch a preestablished anti-RSV Th2 response towards a Th1 response. Critical elements for the optimization of the plasmid constructs included expression of a secretory form of the F protein and the presence of the rabbit beta-globin intron II sequence upstream of the F-encoding sequence. In addition, anti-F systemic immune response profile could be modulated by the route of DNA-F delivery: intramuscular immunization resulted in balanced responses, whereas intradermal immunization resulted in a Th2 type of response. Thus, DNA-F immunization may provide a novel and promising RSV vaccination strategy.

Animals↗
PubMed2002

Phage TP901-1 site-specific integrase functions in human cells.

We demonstrate that the site-specific integrase encoded by phage TP901-1 of Lactococcus lactis subsp. cremoris has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in E. coli and determined that for complete reaction in E. coli, the minimal sizes of attB and attP are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.

Bacteriophages↗
PubMed1993

Sindbis virus attachment: isolation and characterization of mutants with impaired binding to vertebrate cells.

Sindbis virus can infect a broad range of insect and vertebrate cell types. The ability to restrict tissue tropism and target virus infection to specific cell types would expand the usefulness of engineered alphaviruses as gene expression vectors. In this study, virus pools derived from libraries of full-length Sindbis virus cDNA clones containing random insertion mutations in the PE2 or E1 virion glycoprotein gene were screened for mutants defective for binding to vertebrate cells. Binding-competent mutants were depleted by serial adsorption to chicken embryo fibroblast (CEF) monolayers at 4 degrees C, and the remaining population was amplified by immune-enhanced infection of P388D1 cells. From the PE2 libraries, 12 candidate mutants showing reduced cytopathic effects on CEF monolayers were isolated and three representative mutants, NB1, NB2, and NB12, were characterized in detail. Insertion mutations for NB1 and NB12 were found near the PE2 cleavage site, whereas the insertion in NB2 occurred between residues 69 and 74 of E2. Although virion assembly and release occurred normally for all three mutants, PE2 cleavage was completely (NB1) or partially (NB12) blocked for the mutants with insertions near the PE2 cleavage site. Both NB1 and NB2 were defective for binding to CEF and BHK-21 cells. Mild trypsin digestion of isolated NB1 virions resulted in PE2 cleavage and partially restored binding to CEF. Besides defective binding, NB1 also exhibited slower CEF penetration kinetics. Consistent with previous work, these results implicate PE2 cleavage and domains in the N-terminal portion of E2 as important determinants of alphavirus binding and penetration. Binding-defective mutants such as NB2, which exhibit normal particle assembly, release, and penetration, may be useful for future efforts to target Sindbis virus infection.

Amino Acid Sequence↗
PubMed2002

Inserting a nuclear targeting signal into a replication-competent Moloney murine leukemia virus affects viral export and is not sufficient for cell cycle-independent infection.

The effects of inserting reported nuclear localization signals (NLSs) into the Moloney murine leukemia virus (Mo-MuLV) integrase (IN) protein, within a replication-competent viral construct, were studied. In contrast to the virus harboring IN fused to the simian virus 40 (SV40) large T antigen NLS (SV40 NLS) (J. A. Seamon, M. Adams, S. Sengupta, and M. J. Roth, Virology 274:412-419, 2000), a codon-modified SV40 NLS was stably expressed during viral propagation. Incorporation of the codon-modified SV40 NLS into IN, however, altered the packaging of the Gag-Pol precursor in the virus; viral particles contained decreased levels of reverse transcriptase (RT) and IN. In addition, the virus showed delayed kinetics of viral DNA synthesis upon infection. A panel of infectious MuLVs containing alternative IN-NLS fusions was generated and assayed for cell cycle-independent infection. Viral infection with the NLS-tagged proteins, however, remained dependent on passage of the cells through mitosis. This finding has direct implications for engineering murine-based retroviral vectors for gene therapy.

Amino Acid Sequence↗
PubMed1989

[From natural to recombinant human erythropoietin. Biotechnological production of recombinant human erythropoietin].

Human erythropoietin (EPO), which occurs only in minute quantities in human plasma, has been isolated from urine of patients and purified to homogeneity. A partial amino acid sequence from tryptic digests of the purified protein was selected for oligodeoxynucleotide probe synthesis. These probes were used to isolate the EPO gene from a human gene bank. The isolated gene was engineered into a eukaryotic expression vector and introduced into chinese hamster ovary cells commonly used for recombinant protein production. A specific amplification of the EPO gene copy number in these cells led to the secretion of milligram quantities of EPO per liter of cell supernatant. This protein has similar characteristics to native urinary EPO, regarding its electrophoretic mobility, glycosylation pattern and biologic activities in vitro and in vivo. A scaled-up industrial process provides recombinant EPO for its clinical use.

Amino Acid Sequence↗
PubMed2006

Self assembly of DNA nanoparticles with polycations for the delivery of genetic materials into cells.

Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: Material improvement of non-viral vectors and their combination with various external physical stimuli. In this study gelatin was selected as a non-viral carrier for DNA. To give a positive charge to gelatin, different extents introduction of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were reacted with gelatin in the presence of a water-soluble carbodiimide. When positively charged gelatin derivatives (Ed, Sd, and Sm) were mixed with negatively charged DNA, a self assembly of DNA nanoparticle (complex) was formed within few minutes through electrostatic interaction. Irrespective of the type of gelatin derivatives, the apparent molecular size of DNA was reduced by increasing the gelatin/DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin/DNA complexes showed the zeta potential of 10-15 mV. The amount of DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative. The cells incubated with the gelatin/DNA complexes exhibited significantly stronger luciferase activities than naked plasmid DNA. This study clearly demonstrates and self-assembled DNA complexes has potential as a gene delivery vechile and are stable to transfer genetic materials to cells.

Animals↗
PubMed2003

Integrated, molecular engineering approaches to develop prostate cancer gene therapy.

Gene therapy is a translational science, with the ultimate goal of cancer gene therapy research being to develop effective and safe treatments for patients. In the new millennium, it is imperative to tailor a therapeutic strategy for a particular disease, based on clinical management issues. The desirable regulatory features and therapeutic strategies need to be fully considered before proceeding with molecular engineering of the gene delivery vector. Issues, such as cell-targeted expression, in vivo monitoring of gene delivery and expression, therapeutic strategies, and vector selection that targets the particular disease stage should be addressed. During the validation phase of the study, an objective evaluation in relevant animal models should determine whether the vector meets the desired specifications. Meeting the predetermined criteria should propel the product towards the clinical phase of evaluation. This review will present the conceptual framework that has been applied to developing an integrated and targeted gene therapy for prostate cancer.

Animals↗
PubMed2001

Optimism is growing as researchers move toward an AIDS vaccine.

Slow to start, the race for an effective AIDS vaccine now has more than 20 contenders, including some novel approaches that incorporate genetic engineering and the use of vectors, such as a canarypox that doesn't cause disease in humans. Researchers and public health officials say they are more optimistic than ever about the possibility of bringing an AIDS vaccine to market.

AIDS Vaccines↗
PubMed2004

Past and present vaccine development strategies for the control of foot-and-mouth disease.

Foot-and-mouth disease (FMD) virus (FMDV) was the first animal virus to be identified. Since then, it has become a model system in animal virology and more information has been obtained about FMDV. The disease causes heavy economic crises in enzootic countries both due to loss of animal health and productivity. The only way of its control in an enzootic area is strict vaccination and restricted animal movement. The first experimental vaccine against FMD was made in 1925 using formaldehyde inactivation of cattle tongue infected with the virus and this approach remained the basic one until late 1940s. Antigenic plurality and continuous co-circulation of different serotypes in a given geographical region and persistence of virus in infected or vaccinated animals make the disease very difficult to control. The latter is solely based upon the application of isolation, slaughter or aphtisation, and vaccination. With the advent of recombinant DNA technology, recombinant protein and/or DNA-based vaccines are being tested in various heterologous systems for development of FMD vaccines. The subunit vaccines, synthetic peptide vaccines, DNA vaccines, cytokine-enhanced DNA vaccines, recombinant empty capsid vaccines, chimeric viral vaccines, genetically engineered attenuated vaccines, recombinant viral vector vaccines, self-replicating genetic vaccines and transgenic plants with expressed FMDV proteins represent the present vaccine development strategies for control of FMD.

Animals↗
PubMed2005

[Protective efficacy of recombinant rotavirus epitope-based vaccine in mice].

OBJECTIVE: To evaluate in vivo immunological protective efficacy and safety of expressed recombinant rotavirus epitopes in mice. METHODS: Using the Flock House virus capsid protein as a vector, three epitopes derived from rotavirus Vp4 amino acid 223-242 [rotavirus epitope A, (REA)], 243-262 [rotavirus epitope B, (REB)], and 234-251 [rotavirus epitope C, (REC)] were genetically engineered on the surface of the vector protein and expressed in pET-3 (E. coli BL21 [DE3]) system into multiple epitopes, REABC, which comprises REA, REB, and REC. Kunming strain mice were inoculated with the recombinant epitopes REABC, and then challenged perorally by cell culture-adapted rotavirus Wa (type G1P1A) and SA11 (type G3P2). Infection syndrome was observed, and virus antigen in stools of mice and serum neutralizing antibody activities were determined and analyzed. RESULTS: The recombinant epitopes REABC significantly induced rotavirus specific neutralyzing antibodies against WA and SA11, reduced virus reproduction, elicitted immune memory in inoculated mice, and protected inoculated mice from challenge by WA or SA11 (P<0.001). CONCLUSION: The recombinant epitopes have high immunological protective efficacy and mild side effects in mice. It may be used as an epitope-based vaccine candidate in human.

Animals↗
PubMed1993

Carbohydrate-binding protein 35. I. Properties of the recombinant polypeptide and the individuality of the domains.

The cDNA clone for carbohydrate-binding protein 35 (CBP35) was engineered into the bacterial expression vector pIN III ompA2, which directs the secretion of the expressed protein into the periplasmic space. Recombinant CBP35 was purified from this system, at a level of approximately 50 mg/liter of bacterial culture. Digestion of recombinant CBP35 with collagenase D, followed by purification using saccharide-specific affinity chromatography yielded a M(r) approximately 16,000 polypeptide, corresponding to the COOH-terminal domain (residues 118-264) of the CBP35 polypeptide. This indicates that the COOH-terminal half of CBP35 contains the carbohydrate recognition domain, consistent with its sequence homology to other S-type lectins. The NH2-terminal domain (residues 1-137) was derived by site-directed mutagenesis of the cDNA, in which stop codons are inserted in place of Gly138 and Gly139, and expression of the mutant cDNA in the same pIN III ompA2 system. The purified NH2-terminal domain failed to bind to saccharide-specific affinity resins. Differential scanning calorimetry of rCBP35 and its individual domains yielded transition temperatures of approximately 39 and approximately 56 degrees C for the NH2- and COOH-terminal domains, respectively. Lactose binding by the COOH-terminal domain shifted the transition temperature to 65 degrees C, whereas sucrose failed to yield the same effect. These results suggest that the individual domains of the CBP35 polypeptide are folded independently.

Amino Acid Sequence↗
PubMedDate not supplied

Rotavirus vaccines.

Encouraging results have been reported from several large trials of tetravalent rhesus rotavirus vaccine, with efficacy of 70-80% against severe disease. A recent Venezuelan study showed similar results to trials in USA and Europe. The vaccine may soon be licensed in USA. It provides the exciting prospect of a strategy to prevent one of the world's major child killers. Other candidate vaccines are under development including human-bovine reassortants, neonatal strains, non-replicating rotaviruses, vector vaccines and other genetically engineered products. Second and third generation rotavirus vaccines are on the horizon. The need for a rotavirus vaccine is well accepted by paediatricians, but public health authorities need to be lobbied. Other issues which need to be addressed include relative importance of non-group A rotaviruses, possible administration with OPV, the influence of breast feeding, and most importantly, cost. It is essential that rotavirus vaccine is somehow made available to all of the world's children, not just those in developed countries.

Child, Preschool↗
PubMed1998

A novel and efficient method for the stable expression of heteromeric ion channels in mammalian cells.

The 5' untranslated leader sequence from the encephalomyocarditis virus was used to engineer bicistronic or tricistronic expression vectors encoding two subunits (P2X2 and P2X3) of an ATP-gated cation channel. Human embryonic kidney (293) and chinese hamster ovary (CHO-K1) cells were transfected with the vector, and stable cell lines were generated by single cell subcloning. Selection was made in a 96-well format on the basis of a sustained increase in intracellular calcium (fluorescence of Fluo3-loaded cells) evoked by the ATP analog alpha beta methylene ATP. A high proportion of transformants expressed heteromeric receptors containing both P2X2 and P2X3 subunits, as evidenced by a nondesensitizing current in response to alpha beta methylene ATP. The method is fast and simple and could be generally useful for the stable expression of heteromultimeric channel proteins.

Adenosine Triphosphate↗