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Results for “Recombinant expression”

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At least 145 records · Page 8Linked to original sources

PubMed1994

Protection against turkey rhinotracheitis pneumovirus (TRTV) induced by a fowlpox virus recombinant expressing the TRTV fusion glycoprotein (F).

A recombinant fowlpox virus was produced which expressed the fusion protein (F) of turkey rhinotracheitis virus (TRTV), a pneumovirus. Turkey poults were vaccinated twice, at an interval of 2 weeks, intramuscularly and by wing web on each occasion, with the recombinant or a control fowlpox virus. Two weeks after the second vaccination the poults were challenged superconjunctivally and intranasally with virulent TRTV. A partially protective immune response was achieved; turkeys vaccinated with the F recombinant showed milder clinical signs and 1000-fold less challenge virus was recovered from the nose and trachea compared with turkeys that had been vaccinated with control fowlpox virus. Expression of the F protein induced antibodies which were detectable both by an ELISA and a virus neutralization test. These results show that the immune responses to the F protein play a major role in protection against TRTV and indicate that recombinant viruses expressing the TRTV F protein have potential as vaccines against TRT.

Animals↗

PubMed1998

cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity.

Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine lambdagt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by125Iodine labeling and SDS-PAGE.

Amino Acid Sequence↗

PubMed2000

[The construction of PQE-ARL-1 recombinant expression plasmid and the preparation and purification of ARL-1 protein].

OBJECTIVE: To further understand the function and biological activity of aldose reductase like-1 (ARL-1), and to provide the basis for the preparation of the specific antibody against ARL-1. METHODS: The ARL-1 cDNA was cloned into procaryotic expression vector PQE-30 and the recombinant ARL-1 expressed in E.coli M(15); then Ni(+)-NTA agarose columns were used to purify the ARL-1 protein. RESULTS: With the digestion of the enzymes, we identified that the ARL-1 gene was inserted into the procaryotic expression vector PQE-30. The expression products of PQE-ARL-1 (human recombinant ARL-1) showed a single protein band on SDS-PAGE. The molecular weight of ARL-1 was approximately 37.7 x 10(3) and the expression level was about 25% of the total bacterial protein. The concentration of ARL-1 protein was about 100mg/L. CONCLUSION: The construction of the recombinant plasmid PQE-ARL-1 and the preparation of the protein ARL-1 have laid a solid foundation for further studying the function of ARL-1 and preparing the specific antibody against ARL-1.

Aldehyde Reductase↗

PubMed1999

Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation.

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC) and represents the best marker for mature DC. Here, we report the cloning of the cDNA encoding mouse CD83 (mCD83) from a murine bone marrow-derived DC (BM-DC) cDNA library. DNA sequence analysis revealed a 196 amino acid protein including a signal peptide of 21 amino acids which shares 63% amino acid identity with hCD83. Using Northern blot analyses, mCD83 mRNA was found to be strongly expressed in mouse BM-DC and its expression was upregulated following stimulation with LPS or TNF-alpha. Transfection experiments using COS-7 cells revealed that mCD83 is glycosylated. Furthermore, the extracellular CD83 domain was recombinantly expressed in Escherichia coli and one-dimensional NMR data strongly support that the protein is structurally folded.

Amino Acid Sequence↗

PubMed1999

Recombinant expression of polymeric IgA: incorporation of J chain and secretory component of human origin.

Mucosal J (joining) chain-expressing IgA immunocytes produce dimeric IgA that is actively transported by the epithelial polymeric Ig receptor (pIgR) to exocrine secretions. Release of secretory IgA (SIgA) occurs by cleavage of the covalently linked pIgR ectodomain, also known as bound secretory component. We have identified the human J-chain cDNA sequence through database screening, and isolated it from B cells for recombinant expression. Co-expression of this cDNA with an alpha heavy chain and a lambda light chain in Chinese hamster ovary (CHO) cells resulted in a mixture of recombinant monomeric and dimeric IgA in culture supernatants. This dimeric IgA was transported by the pIgR-mediated mechanism in vitro. Furthermore, expression of the human pIgR ectodomain together with the dimeric IgA, resulted in production of complete SIgA by the CHO cells. These results demonstrated that co-expression of the necessary polypeptide components allows a single mammalian cell to produce SIgA. Development of production systems for human antigen-specific recombinant SIgA may be important for applications in passive mucosal vaccination.

Amino Acid Sequence↗

PubMed2004

Improved PC1/3 production through recombinant expression in insect cells and larvae.

Protein convertase 1/3 is a serine endoproteinase present in the regulated secretory pathway of endocrine and neuroendocrine cells. It is responsible for the processing of numerous prohormones and proneuropeptides into their biologically active moieties, often following cleavage at pairs of basic residues. The determination of its three-dimensional structure, as well as the understanding of its enzymatic properties, would greatly benefit from the production and availability of large amounts of recombinant enzyme. We report herein improvements in the production of PC1/3 by expressing recombinant mutated forms in both insect cells (Spodoptera frugiperda, Sf9 cells) and larvae (Trichoplusia ni commonly referred to as cabbage looper). On one hand, we deleted the last 135 COOH-terminal residues of mPC1/3 and, on the other hand, we replaced the signal peptide of mPC1/3 by the viral glycoprotein gp67 signal peptide. These modifications were shown to improve markedly (up to 125%) the secretion into the Sf9 cells medium and the amount of enzymatic activity recovered when compared to the original vector. Moreover, intracoelemic injection of the vectors into insect larvae led to the production and purification of enzymatically active enzyme at a level of 30 microg/larva in the case of mPC1/3 and to the production of a high amount of another enzymatically active convertase, PC7. The optimal viral titer for infection of larvae was determined to be 10(6)pfu/ml. Taking into account the purification protocol combined with the ease and efficiency of using larvae, it should now be possible to meet the needs for biochemical and structural studies.

Animals↗

PubMed1996

Influence of chicken genotype on protection against Marek's disease by a herpesvirus of turkeys recombinant expressing the glycoprotein B (gB) of Marek's disease virus.

Two inbred lines of White Leghorn chickens which differ in B-haplotype were immunized at 2 days of age with a thymidine kinase negative (tk-ve) herpesvirus of turkeys (HVT) recombinant expressing the glycoprotein B (gB) gene of Marek's disease virus (MDV) and were challenged 6 days later with 1000 p.f.u. of the highly virulent RB1B strain of MDV. Mock-vaccinated chickens and chickens immunized with a spontaneous tk-ve HVT mutant served as controls. Genetically resistant B21 chickens were protected by immunization with the recombinant as well as by the tk-ve HVT, whereas highly susceptible B13 chickens were partially protected by the recombinant but were not protected by the tk-ve HVT. Rhode Island Red chickens (HPRS RIR), which differ from the White Leghorns at the B locus, were protected by both vaccines but the recombinant conferred a significantly higher level of protection than the tk-ve HVT. The results suggest that the gB gene of MDV serotype 1 has an important role in the induction of protective immunity against highly virulent MDV in genetically susceptible lines of chickens and that vaccinal immunity in White Leghorns might be influenced by the B haplotype.

Animals↗

PubMed1989

Protection of guinea pigs from Lassa fever by vaccinia virus recombinants expressing the nucleoprotein or the envelope glycoproteins of Lassa virus.

A recombinant vaccinia virus that expresses the nucleoprotein gene of Lassa virus (Josiah strain) under the control of the P7.5 promoter was constructed using the lacZ coexpression transfer vector pSC11. Southern blot analysis demonstrated that recombination of the sequences inserted within the thymidine kinase gene of the transfer vector into the HindIII J fragment of vaccina virus genomic DNA occurred properly. A 63-kDa protein identical in electrophoretic mobility to authentic Lassa nucleoprotein was observed in recombinant virus-infected cell lysates. The reactivity of vaccinia-expressed Lassa proteins to a panel of monoclonal antibodies representing multiple epitopes on each of the N, G1, and G2 proteins was determined by indirect immunofluorescence. Lassa proteins expressed in recombinant vaccinia virus-infected cells reacted in a manner indistinguishable from that of the proteins expressed in Lassa virus-infected cells, indicating that there are no significant differences between authentic and recombinant virus-expressed proteins. Vaccine efficacy trials in guinea pigs indicated that both the nucleoprotein and the envelope glycoproteins are capable of eliciting a protective immune response against a lethal dose of Lassa virus. Ninety-four percent of the animals vaccinated with V-LSN, 79% vaccinated with V-LSGPC, and 58% vaccinated with both recombinant viruses survived a Lassa virus challenge in which only 14% of unvaccinated animals and 39% of animals vaccinated with the New York Board of Health (NYBH) strain of vaccinia virus survived. The protection resulting from vaccination with the recombinant virus vaccines did not correlate with the levels of prechallenge serum antibodies, suggesting that a cell-mediated immune response is a critical component of protective immunity to Lassa fever.

Animals↗

PubMed2006

The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter.

BACKGROUND: The yields of soluble recombinant proteins expressed in bacteria are often low due to the tendency of the heterologous proteins to form aggregates. Therefore, aggregation reporters have been envisaged to simplify the comparison among different expression conditions and to speed up the identification of suitable protocols that improve the solubility. The probe we used is composed by an IbpAB promoter specifically activated by protein aggregates fused to a sequence coding the beta-galactosidase, the activity of which becomes, therefore, indicative of the aggregation degree. RESULTS: The collected data show that the probe is reliable in terms of reproducibility inside a range of experimental conditions and faster and more sensitive than the analysis methods based on SDS-PAGE and successive western blot. The beta-galactosidase probe was useful to identify which parameters could influence the aggregation of the model proteins and to set up an optimized expression protocol. The effect of growth temperature, induction modality, co-expression with molecular chaperones and addition of osmolytes on the accumulation of aggregates were evaluated following the beta-galactosidase activity. Interestingly, a significant correlation was observed between estimated decreased aggregation and higher yields of soluble protein. We also compared a set of expression vectors with various regulative features and found that the single characteristics, like promoter, copy number or polymerase, were not relevant for controlling the recombinant protein aggregation whilst the crucial factor resulted being the total expression rate of the system. CONCLUSION: The aggregation reporter used in our experiments represents a useful tool to evaluate the different factors that can be modulated to optimize a recombinant expression protocol. Furthermore, the rapid estimation of the aggregation degree enables to discriminate this from other causes responsible for scarce recombinant yields.

Journal Article↗

PubMed2006

SARS-CoV spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus.

A vaccine for severe acute respiratory syndrome (SARS) is being intensively pursued against its re-emergence. We generated a SARS coronavirus (SARS-CoV) spike protein-expressing recombinant vaccinia virus (RVV-S) using highly attenuated strain LC16m8. Intradermal administration of RVV-S into rabbits induced neutralizing (NT) antibodies against SARS-CoV 1 week after administration and the NT titer reached 1:1000 after boost immunization with RVV-S. Significantly, NT antibodies against SARS-CoV were induced by administration of RVV-S to rabbits that had been pre-immunized with LC16m8. RVV-S can induce NT antibodies against SARS-CoV despite the presence of NT antibodies against VV. These results suggest that RVV-S may be a powerful SARS vaccine, including in patients previously immunized with the smallpox vaccine.

Animals↗

PubMed2003

Prolonged exposure to gamma-aminobutyric acid up-regulates stably expressed recombinant alpha 1 beta 2 gamma 2s GABAA receptors.

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABA(A) receptors following their prolonged exposure to drugs. Exposure (48 and/or 96 h) of human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors for gamma-aminobutyric (GABA, 1 mM) and muscimol (100 microM), but not for diazepam (1 microM), enhanced the maximum number (B(max)) of [3H]flunitrazepam binding sites without affecting their affinity (K(d)). The GABA-induced enhancement in B(max) was reduced by the GABA receptor antagonist, bicuculline (100 microM), and by cycloheximide (10 microl/ml), a protein synthesis inhibitor. GABA (100 microM) enhanced the affinity of [3H]flunitrazepam binding to vehicle- and GABA-pretreated, but not to diazepam-pretreated, HEK 293 cells. The results suggest that chronic GABA treatment up-regulates stably expressed GABA(A) receptors, presumably by stimulating their synthesis. Unlike chronic diazepam, which produced functional uncoupling of GABA and benzodiazepine binding sites, chronic GABA failed to produce this effect.

Cell Line↗

PubMed1991

Assembly of empty capsids by using baculovirus recombinants expressing human parvovirus B19 structural proteins.

Empty parvovirus B19 capsids were isolated from insect cells infected with a recombinant baculovirus expressing parvovirus B19 VP2 alone and also with a double-recombinant baculovirus expressing both VP1 and VP2. That VP2 alone can assemble to form capsids is a phenomenon not previously observed in parvoviruses. The stoichiometry of the capsids containing both VP1 and VP2 was similar to that previously observed in parvovirus B19-infected cells. The capsids were similar to native capsids in size and appearance, and their antigenicity was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assay with B19-specific antibodies.

Animals↗

PubMed1996

Effects of long-term treatment with the allosteric enhancer, PD81,723, on Chinese hamster ovary cells expressing recombinant human A1 adenosine receptors.

In this study, desensitization and down-regulation of A1 adenosine receptors (A1AR) by the allosteric enhancer PD81,723 (PD) and by N6-cyclopentyladenosine (CPA) were investigated after 24-hr pretreatment of CHO-K1 cells stably expressing recombinant human A1AR. Pretreatment with 20 microM PD and 10 microM CPA caused a 1.5- and 4.0-fold, respectively, desensitization (reduced potency of CPA to lower cAMP). Pretreatment with PD and/or CPA did not modify the acute effect of PD to increase (5-fold) the potency of CPA. Radioligand binding was used to measure receptor down-regulation in cell membranes and in intact cells. Pretreatment of cells with PD had no effect on the number of membrane binding sites for the agonist [125I] N6-(3-iodo-4-aminobenzyl) adenosine or for the antagonist, [3H]8-cyclopentyl-1,3-dipropylxanthine, but the binding of these radioligands to intact cells was modestly reduced (20-37%), possibly reflecting an effect of pretreatment on receptor subcellular distribution. Pretreatment of cells with CPA produced large ( > 40%) reductions in the binding of radioligands to both membranes and intact cells. Pretreatment of cells with CPA also increased the number of presumed internalized receptors measured as [3H]8-cyclopentyl-1,3-dipropylxanthine binding sites in intact cells insensitive to blockade by the charged antagonist 8-sulfophenyltheophylline. The relatively small degree of functional desensitization and down-regulation of A1AR caused by long term exposure of cells to PD is considered to be encouraging in terms of the therapeutic potential of the allosteric enhancer class of compounds.

Adenosine↗

PubMed1997

Recombinant expression of the antimicrobial peptide polyphemusin and its activity against the protozoan oyster pathogen Perkinsus marinus.

Polyphemusin is a broad-spectrum antimicrobial peptide isolated from hemocytes of the North American horseshoe crab Limulus polyphemus. To date the polyphemusin used for scientific analyses has been purified from the natural materials or obtained by chemical synthesis. We report here the recombinant expression in Escherichia coli, and subsequent purification, of a polyphemusin analogue (rLim1). To prevent toxicity of the antimicrobial peptide in the highly susceptible E. coli host, we used a carboxy-terminal fusion protein cloning strategy provided by a maltose-binding protein (MBP) gene fusion system (New England Biolabs). Antimicrobial activity of recombinant polyphemusin was similar to that seen with amidated native polyphemusin peptide. When rLim1 was tested for antibiotic activity against the apicomplexan protozoan oyster pathogen Perkinsus marinus, complete inhibition was observed at 12 micrograms/ml, and partial inhibition at 8 micrograms/ml.

ATP-Binding Cassette Transporters↗

PubMed2000

Cloning and recombinant expression of a structurally novel human secreted phospholipase A2.

Mammals contain a diverse set of secreted phospholipases A(2) (sPLA(2)s) that liberate arachidonic acid from phospholipids for the production of eicosanoids and exert a variety of physiological and pathological effects. We report the cloning, recombinant expression, and kinetic properties of a novel human sPLA(2) that defines a new structural class of sPLA(2)s called group XII. The human group XII (hGXII) cDNA contains a putative signal peptide of 22 residues followed by a mature protein of 167 amino acids that displays homology to all known sPLA(2)s only over a short stretch of amino acids in the active site region. Northern blot and reverse transcription-polymerase chain reaction analyses show that the tissue distribution of hGXII is distinct from the other human sPLA(2)s with strong expression in heart, skeletal muscle, kidney, and pancreas and weaker expression in brain, liver, small intestine, lung, placenta, ovaries, testis, and prostate. Catalytically active hGXII was produced in Escherichia coli and shown to be Ca(2+)-dependent despite the fact that it is predicted to have an unusual Ca(2+)-binding loop. Similar to the previously characterized mouse group IIE sPLA(2)s, the specific activity of hGXII is low in comparison to that of other mammalian sPLA(2), suggesting that hGXII could have novel functions that are independent of its phospholipase A(2) activity.

Amino Acid Sequence↗

PubMed2006

Recombinant expression of Toluene o-Xylene Monooxygenase (ToMO) from Pseudomonas stutzeri OX1 in the marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

The psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125, isolated from Antarctic seawater, was used as recipient for a biodegradative gene of the mesophilic Pseudomonas stutzeri OX1. tou cluster, coding for Toluene o-Xylene Monooxygenase (ToMO), was successfully cloned and expressed into a "cold expression" vector. Apparent catalytic parameters of the recombinant microorganisms on three different substrates were determined and compared with those exhibited by Escherichia coli recombinant cells expressing ToMO. Production of a catalytically efficient TAC/tou microorganism supports the possibility of developing specific degradative capabilities for the bioremediation of chemically contaminated marine environments and of industrial effluents characterised by low temperatures.

Antarctic Regions↗

PubMed1998

Structure, genomic localization and recombinant expression of the mouse 6-pyruvoyl-tetrahydropterin synthase gene.

The 6-pyruvoyl-tetrahydropterin synthase (PTPS) is the second enzyme in the biosynthetic pathway from GTP to tetrahydrobiopterin (BH4). BH4 is an essential cofactor of NO synthases and aromatic amino acid hydroxylases, the latter being responsible for hepatic phenylalanine degradation and monoamine neurotransmitter biosynthesis. BH4 deficiency due to autosomal recessive mutations in the human gene for PTPS leads to a broad range of phenotypes ranging from mild hyperphenylalaninemia to high phenylalanine levels concomitant with neurotransmitter depletion. An animal model to study PTPS deficiency is thus desired to investigate the molecular basis of the disease and its variability. Here, we report on the isolation and recombinant expression of the mouse PTPS gene, Pts. It is located on chromosome 9C-D and contains six exons with an open reading frame of 144 codons. The derived protein monomer has a molecular mass of 16187 Da and shows 82% and 93% identity to its human and rat counterparts, respectively. The mouse PTPS was expressed in bacterial cells and purified to homogeneity. The kinetic properties of the recombinant protein, apparent Km of approximately 10 microM and k(cat) of 0.27 s(-1), were similar to the native mouse enzyme in liver and brain extracts, and to the corresponding human and rat PTPS.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of soluble forms of human and rat stem cell factor recombinantly expressed by Escherichia coli and by Chinese hamster ovary cells.

Stem cell factor (SCF) is a novel, early-acting hematopoietic factor. It was isolated from the medium of a rat cell line in a soluble, processed form (Zsebo et al., 1990, Cell 63, 195). The cloned human and rat genes encode the soluble form plus additional C-terminal amino acids including a hydrophobic transmembrane domain (Martin et al., 1990, Cell 63, 203). We have recombinantly expressed forms of human and rat SCF corresponding to the soluble, processed form in Escherichia coli and in Chinese hamster ovary (CHO) cells. After expression in E. coli, folding and oxidation of the SCF polypeptides are required. The SCFs expressed in CHO cells are secreted into the medium in active state and, like the natural SCF, are glycosylated. Purification of the recombinant SCFs is described. Biological and biochemical characterization includes activity toward responsive human and mouse cell lines, N-terminal amino acid sequences, disulfide bond linkages, and sites of glycosylation.

Amino Acid Sequence↗