SEARCH · PubMed Health

Results for “Recombinant expression”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources

PubMed1998

Influence of anesthetics on endogenous and recombinantly expressed G protein-coupled receptors in the Xenopus oocyte.

1. The oocyte of the African clawed toad (Xenopus laevis) offers a reliable, sensitive and disease resistant system to investigate recombinantly and endogenously expressed Ca2+ signaling G protein-coupled receptors and their intracellular signaling pathways. 2. To study receptor induced Ca2+ release, two-electrode voltage clamping can quantify a Ca2+-activated transmembrane Cl- current. Intracellular steps of the signaling pathway can be inhibited by injections of EDTA or heparin into the oocyte. Components of the intracellular pathway can be activated directly by GTPgammaS or IP3 injection. 3. We have investigated the effects of volatile, local and i.v. anesthetics on the signaling properties of the endogenous lysophosphatidate receptor and on mammalian receptors expressed recombinantly by intracellular injection of the encoding mRNA or cDNA. A number of receptors are sensitive to these anesthetics. Anesthetics interact with muscarinic, thromboxane A2 and lysophosphatidate signaling. 4. Investigations of the intracellular pathways revealed that the receptor or the receptor-G protein coupling is affected primarily and that mechanisms further downstream are not influenced by the various types of anesthetics.

Anesthetics↗

PubMed2000

Humoral immune response of BALB/c mice to a vaccinia virus recombinant expressing Brucella abortus GroEL does not correlate with protection against a B. abortus challenge.

This work is a part of an ongoing effort to develop vaccinia virus recombinants expressing various Brucella abortus proteins. The B. abortus groEL gene encoding the antigenic heat shock protein GroEL was subcloned into vaccinia virus via homologous recombination and expression confirmed by Western blotting. Female BALB/c mice inoculated with recombinant vaccinia virus/GroEL produced GroEL and vaccinia virus specific antibodies. Mice were challenged 8 weeks post-inoculation with virulent B. abortus strain 2308 and protection measured by the rate of clearance of live Brucella from spleens. Although induction of specific immune response to GroEL and vaccinia virus was demonstrated by the appearance of antibodies in mice, no significant level of protection was demonstrable.

Animals↗

PubMed2005

Recombinant expression of bovine LFA-1 and characterization of its role as a receptor for Mannheimia haemolytica leukotoxin.

Mannheimia (Pasteurella) haemolytica leukotoxin (LktA) is the primary virulence factor contributing to the pathogenesis of lung injury in bovine pneumonic mannheimiosis (BPM), a disease which causes major economic loss to the US cattle industry annually. Recent work from our laboratory using an antibody-based approach has shown that LktA binds to bovine LFA-1 in target cells. While this study suggests that LFA-1 might be a specific receptor, it remains to be conclusively shown that LFA-1 is sufficient to induce susceptibility to LktA. It was of interest to determine if functionally active bovine LFA-1 could be reconstituted on a LFA-1 negative cell line and reconstitute susceptibility to LktA. Here, we report the successful recombinant expression of bovine LFA-1 on the cell surface of the human erythroleukemic K562 cell line. The BoLFA-1 transductant expresses bovine CD18 and CD11a as a heterodimer. We found that LktA binds to both the CD18 and CD11a subunits of BoLFA-1 cells. Exposure of BoLFA-1 cells to LktA, induced tyrosine phosphorylation of the CD18 tail, elevation of intracellular calcium, and cytolysis. This is the first report on recombinant expression of functionally active bovine LFA-1 by transduction into an LktA-non-susceptible human cell line.

Animals↗

PubMed2002

Immunofluorescence technique using HeLa cells expressing recombinant nucleoprotein for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus.

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.

Animals↗

PubMedDate not supplied

Cloning and recombinant expression of a barred sand bass (Paralabrax nebulifer) cDNA. The encoded protein displays structural homology and immunological crossreactivity to human complement/cofactor related plasma proteins.

A new cofactor related cDNA in the bony fish Paralablax nebulifer, (barred sand bass) was isolated from a sand bass liver cDNA library. The clone (c71) is 1040 bp in size and the predicted translation product of 204 amino acids contains a hydrophobic signal peptide, which is followed by a region of three short consensus repeats (SCRs). The three SCRs display high homology to SCRs of the 110 kDa chain of the sand bass plasma cofactor protein, and to a lesser degree to human complement factor H related protein 3 (FHR-3) and to human factor H. Recombinant expression of the c71 cDNA in the baculovirus system shows a product of an apparent molecular mass of 27 kDa, which is secreted and glycosylated. It also contains a His-tag for purification purposes. Removal of the His-tag yields a 24 kDa protein, and deglycosylation further reduces the molecular mass to 21 kDa. This size is in agreement with the calculated molecular mass based on amino acid composition. The sand bass SBCFR-1 protein is immunologically related to the human complement proteins, factor H and factor H-related protein 3. The recombinantly expressed protein reacted with antisera against the human FHR-3 protein and SCRs 19-20 of human factor H. The presence of SCR-containing proteins in sand bass plasma and their structural and immunological homology to human FHR-3 and factor H suggests for a common function between these evolutionary related proteins.

Amino Acid Sequence↗

PubMed2004

Intein-mediated purification of a recombinantly expressed peptide.

A 26 amino acid peptide has successfully been purified via recombinant expression as an intein fusion protein accompanied by cleavage without the need for any exogenous proteases or cofactors, thus offering a practical, inexpensive approach to produce isotopically labelled peptides.

Hydrogen-Ion Concentration↗

PubMed1992

Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein.

Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.

Animals↗

PubMed1992

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.

Animals↗

PubMed1992

Generation of monoclonal antibodies to human lymphocyte cell surface antigens using insect cells expressing recombinant proteins.

We have expressed human CD40 and human B7 in insect cells using the baculovirus expression system and have used these insect cells to immunize mice for the generation of monoclonal antibodies. We demonstrate here that specific monoclonal antibodies to human CD40 and human B7 were obtained using this approach. One significant advantage of this method is that immunizing mice with insect cells did not evoke an immune response to human cells and, therefore, EBV-transformed human B cells could be used to screen for specific antibody production by the hybridoma clones.

Amino Acid Sequence↗

PubMed2003

Recombinant expression and characterization of a novel fibronectin isoform expressed in cartilaginous tissues.

A novel fibronectin (FN) isoform lacking the segment from IIICS (type III connecting segment) through the I-10 module is expressed predominantly in normal cartilaginous tissues. We expressed and purified recombinant cartilage-type FN using a mammalian expression system and characterized its molecular and biological properties. Although FNs have been shown to be secreted as disulfide-bonded dimers, cartilage-type FN was secreted mainly as a monomer. It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin alpha5beta1, although it was more active than plasma-type FN in binding to chondroitin sulfate E. When added exogenously, cartilage-type FN was poorly assembled into the fibrillar FN matrix, mostly because of its monomeric structure. Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing.

Animals↗

PubMed1999

[Immune responses to recombinant expression plasmid encoding hepatitis C virus core antigen].

OBJECTIVE: To study the feasibility of DNA immunization with recombinant expression plasmid containing hepatitis C virus (HCV) core gene against HCV infection. METHODS: The recombinant plasmid containing HCV C gene was constructed and expressed transiently by using lipofectamine in the mouse SP2/0 cells. After appraisal and purification, these plasmid and control DNA were directly injected intramuscularly into BALB/c mice, HCV C specific antibody in the sera was checked with ELISA. Proliferative responses of spleen cells from experimental and control mice were determined by (3)H thymidine incorporation and cytotoxicity T lymphocyte (CTL) activity was measured by standard 4-h (51)Cr release assays. RESULTS: All immunized mice developed anti-HCV core antibodies and this antibody was dose and immune number dependent. Lymphoproliferative responses for HCV core antigen were higher in immunized mice than in control mice (P < 0.05), stimulation indices (SI) were 2.42 - 6.10. CTLs might induce approximately 63.13% lysis of pcDNA HCV C transfected SP2/0 target cells. CONCLUSION: It is suggested that HCV C gene immunization is a promising method of vaccination against HCV infection.

Animals↗

PubMed2000

Cloning and recombinant expression of human group IIF-secreted phospholipase A(2).

Mammalian-secreted phospholipases A(2) (sPLA(2)) form a diverse family of at least nine enzymes that hydrolyze phospholipids to release free fatty acids and lysophospholipids. We report here the cloning and characterization of human group IIF sPLA(2) (hGIIF sPLA(2)). The full-length cDNA codes for a signal peptide of 20 amino acid followed by a mature protein of 148 amino acids containing all of the structural features of catalytically active group II sPLA(2)s. hGIIF sPLA(2) gene is located on chromosome 1 and lies within a sPLA(2) gene cluster of about 300 kbp that also contains the genes for group IIA, IIC, IID, IIE, and V sPLA(2)s. In adult tissues, hGIIF is highly expressed in placenta, testis, thymus, liver, and kidney. Finally, recombinant expression of hGIIF sPLA(2) in Escherichia coli shows that the enzyme is Ca(2+)-dependent, maximally active at pH 7-8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference.

Amino Acid Sequence↗

PubMed2004

Ca2+ transport in mitochondria from yeast expressing recombinant aequorin.

We have expressed aequorin in mitochondria of the yeast Saccharomyces cerevisiae and characterized the resulting strain with respect to mitochondrial Ca(2+) transport in vivo and in vitro. When intact cells are suspended in water containing 1.4 mM ethanol and 14 mM CaCl(2), the matrix free Ca(2+) concentration is 200 nM, similar to the values expected in cytoplasm. Addition of ionophore ETH 129 allows an active accumulation of Ca(2+) and promptly increases the value to 1.2 microM. Elevated Ca(2+) concentrations are maintained for periods of 6 min or longer under these conditions. Isolated yeast mitochondria oxidizing ethanol also accumulate Ca(2+) when ETH 129 is present, but the cation is not retained depending on the medium conditions. This finding confirms the presence of a Ca(2+) release mechanism that requires free fatty acids as previously described [P.C. Bradshaw et al. (2001) J. Biol. Chem. 276, 40502-40509]. When a respiratory substrate is not present, Ca(2+) enters and leaves yeast mitochondria slowly, at a specific activity near 0.2 nmol/min/mg protein. Transport under these conditions equilibrates the internal and external concentrations of Ca(2+) and is not affected by ruthenium red, uncouplers, or ionophores that perturb transmembrane gradients of charge and pH. This activity displays sigmoid kinetics and a K(1/2) value for Ca(2+) that is near to 900 nM, in the absence of ethanol or when it is present. It is furthermore shown that the activity coefficient of Ca(2+) in yeast mitochondria is a function of the matrix Ca(2+) content and is substantially larger than that in mammalian mitochondria. Characteristics of the aequorin-expressing strain appear suitable for its use in expression-based methods directed at cloning Ca(2+) transporters from mammalian mitochondria and for further examining the interrelationships between mitochondrial and cytoplasmic Ca(2+) in yeast.

Aequorin↗

PubMed1996

Modulation of 45Ca2+ influx into cells stably expressing recombinant human NMDA receptors by ligands acting at distinct recognition sites.

A 45Ca2+ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N-methyl-D-aspartate (NMDA) receptors. Inhibition of glutamate-stimulated 45Ca2+ influx by six glycine-site antagonists and inhibition of glycine-stimulated 45Ca2+ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated 45Ca2+ influx for recombinant NMDA receptors, inhibiting and stimulating 45Ca2+ influx into cells expressing NR1a/NR2A receptors (IC50 = 408 microM) and NR1a/NR2B receptors (EC50 = 37.3 microM), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC50 = 0.099 microM) compared with NR1a/NR2A receptors (IC50 = 164 microM). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg2+ and Zn2+ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of 45Ca2+ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.

Animals↗

PubMed2000

Cloning and recombinant expression of the La RNA-binding protein from Trypanosoma brucei.

We report the isolation, cloning and recombinant expression of a Trypanosoma brucei homolog of the La RNA-binding protein. Based on peptide sequence information we have isolated a cDNA clone which encodes a protein of 335 amino acids with a predicted molecular weight of 37.7 kDa. The amino acid sequence fits the domain structure of known La proteins and contains a putative ATP-binding site located in the COOH-terminal domain. The cDNA was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and the recombinant protein displayed RNA-binding activity in an electrophoretic mobility shift assay.

Amino Acid Sequence↗

PubMed2003

Recombinant expression, purification, and comparative characterization of torsinA and its torsion dystonia-associated variant Delta E-torsinA.

Early-onset torsion dystonia is an autosomal dominant movement disorder that has been linked to the deletion of one of a pair of glutamic acid residues in the protein torsinA (E(302/303); DeltaE-torsinA). In transfected cells, DeltaE-torsinA exhibits similar biochemical properties to wild type (WT)-torsinA, but displays a distinct subcellular localization. Primary structural analysis of torsinA suggests that this protein is a membrane-associated member of the AAA family of ATP-binding proteins. However, to date, neither WT- nor DeltaE-torsinA has been obtained in sufficient quantity and purity to permit detailed biochemical and biophysical characterization. Here, we report a baculovirus expression system that provides milligram quantities of purified torsin proteins. Recombinant WT- and DeltaE-torsinA were found to be membrane-associated glycoproteins that required detergents for solubilization and purification. Analysis of the biophysical properties of WT- and DeltaE-torsinA indicated that both proteins were folded monomers in solution that exhibited equivalent denaturation behaviors under thermal and chaotropic (guanidinium chloride) stress. Additionally, both forms of torsinA were found to display ATPase activity with similar k(cat) and K(m) values. Collectively, these data reveal that torsinA is a membrane-associated ATPase and indicate that the DeltaE(302/303) dystonia-associated mutation in this protein does not cause gross changes in its catalytic or structural properties. These findings are consistent with a disease mechanism in which DeltaE-torsinA promotes dystonia through a gain rather than loss of function. The recombinant expression system for torsinA proteins described herein should facilitate further biochemical and structural investigations to test this hypothesis.

Adenosine Triphosphatases↗

PubMed1991

Introduction of vascular smooth muscle cells expressing recombinant genes in vivo.

Vascular smooth muscle cells contribute to the formation of atherosclerotic plaques by proliferating in response to vascular injury and releasing growth-promoting factors. Because their autocrine and paracrine effects are not fully understood, expression of such growth factor genes in specific cell types in vivo would help to determine their mechanism of action. We describe a method to transfer vascular smooth muscle cells expressing recombinant gene products to localized segments of the arterial wall. Vascular smooth muscle cells from the inbred Yucatan minipig were infected in vitro with an amphotropic, replication-defective retrovirus transducing the gene for Escherichia coli beta-galactosidase. Vascular smooth muscle cells expressing this recombinant gene were implanted, using a catheter, into denuded iliofemoral artery segments of pigs in vivo. These arteries subsequently demonstrated beta-galactosidase activity in cells of the intima and media. This method, which provides for the introduction of genetically modified smooth muscle cells, can be used to define the biological effects of recombinant genes in the vessel wall and potentially to provide alternative treatments of vascular diseases.

Animals↗

PubMed2000

Carbohydrate structures of soluble human L-selectin recombinantly expressed in baby-hamster kidney cells.

A soluble form of L-selectin was recombinantly produced, which might be an effective therapeutic agent in inflammatory disorders, acting as an inhibitor for leucocyte endothelium adhesion. In the present study the oligosaccharide structures of soluble human L-selectin, recombinantly expressed in baby-hamster kidney cells, were determined. The N-linked glycans were enzymically released and fluorescently labelled with 2-aminobenzamide. Sialylation of the N-glycans was analysed by anion-exchange chromatography followed by rechromatography of the resulting fractions on amino-phase HPLC after release of the sialic acid residues. Desialylated oligosaccharides were separated using two-dimensional HPLC and characterized by digestion with exoglycosidases and MS. More than 30 oligosaccharide structures representing at least 95% of the overall glycosylation of this protein were determined. The results revealed that recombinant soluble human L-selectin carries bi-, tri- and tetra-antennary sugar chains, which are fucosylated on the innermost residue of N-acetylglucosamine. The number of sialic acid residues linked to these glycans ranges from 0 (neutral glycans) to 4 (tetrasialylated oligosaccharides). The sialic acid is found exclusively in the alpha 2-3 linkage to galactose. In addition to the main glycans, different minor structures containing terminal N-acetylgalactosamine, or the H (O) blood-group determinant were also identified. O-Glycosylation of mucin-type sugar chains was not detected in recombinant soluble human L-selectin.

Animals↗