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At least 577 records · Page 32Linked to original sources
PubMed1998

Characterization of a recombinant human calicivirus capsid protein expressed in mammalian cells.

The capsid protein of the Hawaii strain of human calicivirus was expressed in the transient MVA/bacteriophage T7 polymerase hybrid expression system in order to examine its processing in mammalian cells. Selected amino acid modifications (an insertion, deletion, and substitution) at the predicted amino terminus of the capsid protein as well as the presence or absence of the ORF3 gene were examined for their effect on capsid expression. The protein was expressed efficiently in cell lines derived from three different species, with most of the expressed protein remaining localized within the cells. There was no evidence for N-linked glycosylation or myristylation of the 57 kDa capsid protein. Hawaii virus-like particles (HV VLPs), efficiently produced in the baculovirus expression system, were not observed in this expression system under the conditions in this study.

Animals↗
PubMed1996

Defective HSV-1 vector expressing BDNF in auditory ganglia elicits neurite outgrowth: model for treatment of neuron loss following cochlear degeneration.

The neurotrophins are a family of growth factors that play an important role in the development and maintenance of the nervous system. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that appears to participate in the maturation and function of mammalian auditory neurons. Forms of deafness due to varied injurious stimuli that are amenable to treatment with implantable prosthetic devices require the survival of these BDNF-responsive auditory neurons for effective outcome. To evaluate the feasibility of developing a gene therapy for deafness that may be used in conjunction with a prosthetic device, we constructed replication-defective herpes simplex virus (HSV) amplicon vectors that carry the human BDNF cDNA. Using these vectors, HSVbdnf and HSVbdnflac (expresses BDNF and Escherichia coli beta-galactosidase), we evaluated the expression and biological activity in established cell lines and explant cultures prepared from spiral ganglia of the murine ear. Gene transfer with HSVbdnf resulted in the efficient expression of human BDNF mRNA in murine fibroblasts. Using two BDNF-responsive cell lines, PC12trkB and MG87trkB, we demonstrate efficient secretion of biologically active BDNF. Finally, transduction of explanted spiral ganglia with HSVbdnflac elicited robust neuritic process outgrowth comparable to exogenously added BDNF. Overall, these data demonstrate that HSV vectors can efficiently transfer and express the BDNF gene in many cell types, including auditory neurons. Moreover, they suggest that similar vectors may be used to express the neurotrophin in auditory neurons in vivo and perhaps as adjunctive gene therapy for deafness.

Animals↗
PubMed2005

The polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation.

Mammalian host factors required for efficient viral gene expression and propagation have been often recalcitrant to genetic analysis. A case in point is the function of cellular factors that trans-activate internal ribosomal entry site (IRES)-driven translation, which is operative in many positive-stranded RNA viruses, including all picornaviruses. These IRES trans-acting factors have been elegantly studied in vitro, but their in vivo importance for viral gene expression and propagation has not been widely confirmed experimentally. Here we use RNA interference to deplete mammalian cells of one such factor, the polypyrimidine tract binding protein, and test its requirement in picornavirus gene expression and propagation. Depletion of the polypyrimidine tract binding protein resulted in a marked delay of particle propagation and significantly decreased synthesis and accumulation of viral proteins of poliovirus and encephalomyocarditis virus. These effects could be partially restored by expression of an RNA interference-resistant exogenous polypyrimidine tract binding protein. These data indicate a critical role for the polypyrimidine tract binding protein in picornavirus gene expression and strongly suggest a requirement for efficient IRES-dependent translation.

DNA-Binding Proteins↗
PubMed2005

Efficient Tet-dependent expression of human factor IX in vivo by a new self-regulating lentiviral vector.

Regulation of gene expression represents a long-sought goal of gene therapy. However, most viral vectors pose constraints on the incorporation of drug-dependent transcriptional regulatory systems. Here, by optimizing the design of self-regulating lentiviral vectors based on the tetracycline system, we have been able to overcome the limitations of previously reported constructs and to reach both robust expression and efficient regulation from a single vector. The improved performance allows us to report for the first time effective long-term in vivo regulation of a human clotting Factor IX (hF.IX) transgene upon systemic administration of a single vector to SCID mice. We showed that hF.IX expression in the plasma could be expressed to therapeutically significant concentrations, adjusted to different set levels by varying the tetracycline dose, rapidly turned off and on, and completely recovered after each treatment cycle. The new vector design was versatile, as it successfully incorporated a tissue-specific promoter that selectively targeted regulated expression to hepatocytes. Robust transgene expression in the systemic circulation coupled to the ability to switch off and even adjust the expression level may open the way to safer gene-based delivery of therapeutics.

Animals↗
PubMed1992

Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3, following their expression in Escherichia coli as fusion proteins.

The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn. The latter two residues provide a unique hydroxylamine-sensitive link between [Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity. This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I respectively. Production of milligram quantities of IGF-I peptide was readily achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis. [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.

Amino Acid Sequence↗
PubMed2004

[Cloning, expression and identification of human survivin in E. coli].

OBJECTIVE: To efficiently express and identify recombinant human survivin in E. coli. METHODS: Survivin cDNA was amplified by reverse transcriptional (RT)-PCR and cloned into the prokaryotic expression vector pBV220, followed by expression of the recombinant plasmid in E.coli strain BL21 (Gold). To obtain survivin protein, DEAE-Sepharose Fast-Flow ion exchange chromatography and Sephacryl S-200 gel filtration were performed. Western blot analysis was used for detecting the expressed product. RESULTS: Survivin protein was expressed in E.coli in the form of inclusion body at the expression level over 30% of the total cell protein. After ion exchange chromatography and gel filtration, the recombinant protein reached a purity over 95% and exhibited specific reaction with mouse anti-human antibody. CONCLUSION: Survivin protein with high purity can be obtained by the method described above to facilitate further study of the anti-apoptosis function of survivin.

Cloning, Molecular↗
PubMed2000

Highly efficient transduction and expression of cytokine genes in human tumor cells by means of autonomous parvovirus vectors; generation of antitumor responses in recipient mice.

The possible use of recombinant autonomous parvoviruses as vectors to efficiently express therapeutic cytokines in human tumor cells was evaluated in vitro and in vivo. The parvovirus H1 was used to generate recombinant viruses (rH1) that carried transgenes encoding either human interleukin 2 (IL-2) or monocyte chemotactic protein 1 (MCP-1), in replacement of part of the capsid genes. Such rH11 viruses have been shown to retain in vitro the intrinsic oncotropic properties of the parental virus. On infection with the recombinant viruses at an input multiplicity of 1 replication unit (RU) per cell, HeLa cultures were induced to release 4-10 microg of cytokine per 10(6) cells over a period of 5 days. The expression of the rH1-transduced human cytokine/chemokine could also be detected in tumor material recovered from nude mice that had been subcutaneously engrafted with in vitro-infected HeLa cells. The formation of tumors from HeLa xenografts was reduced by 90% compared with wild-type or mock-infected cells as a result of cells preinfected with IL2-expressing virus at an input multiplicity as low as 1 RU per cell. Tumors arising from HeLa cells infected with transgene-free or MCP1-expressing vectors or with wild-type H1 virus were not rejected at this virus dose. Tumors infected with rH1/IL-2 virus displayed markers indicative of their infiltration with NK cells in which the cytocidal program was activated, whereas little NK activity was detected in wild-type virus or mock-infected tumors. Altogether, these data show that the IL-2 expressing H1 vector was a more potent antineoplastic agent than the parental virus, and point to the possible application of recombinant autonomous parvoviruses toward therapy of some human tumors.

Animals↗
PubMed1992

Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits.

The glycoprotein (GP) Ib-IX complex of the platelet plasma membrane mediates the adhesion of platelets to damaged blood vessel wall. The complex is composed of three membrane-spanning polypeptides, GP Ib alpha, GP Ib beta, and GP IX, all of which are absent from the platelets of patients with the hereditary bleeding disorder Bernard-Soulier syndrome. In this study we report stable expression of the recombinant receptor in three cell lines and demonstrate that the three subunits of the complex are necessary for its efficient expression on the plasma membrane. The expressed complex associates with the cytoskeleton of the transfected cells through an interaction with actin-binding protein and binds its ligand, von Willebrand factor. These data suggest that the lack of plasma membrane GP Ib-IX complex in the Bernard-Soulier syndrome could potentially arise from mutations affecting any one of its three subunits.

Animals↗
PubMed1993

The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120 (ENV), is required in large quantities for immunological studies and as a potential vaccine component. We have expressed the DNA encoding gp120 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. The native gene was found to contain a sequence which resembled a Saccharomyces cerevisiae polyadenylation consensus and acted as a premature polyadenylation site in P. pastoris, resulting in the production of truncated mRNA. As full-length mRNA was produced in S. cerevisiae, this indicates differences in mRNA 3'-end formation between the two yeasts. Inactivation of this site by site-directed mutagenesis revealed several additional fortuitous polyadenylation sites within the gene. We have designed and constructed a 69%-synthetic gene with increased G + C content which overcomes this transcriptional problem, giving rise to full-length mRNA. High levels of intracellular, insoluble, unglycosylated ENV were produced [1.25 mg/ml in high-density (2 x 10(10) cells per ml) fermentations]. ENV also was secreted from P. pastoris using the S. cerevisiae alpha-factor prepro secretion leader and the S. cerevisiae invertase signal sequence. However, a high proportion of the secreted product was found to be hyperglycosylated, in contrast to other foreign proteins secreted from P. pastoris. There also was substantial proteolytic degradation, but this was minimized by maintaining a low pH on induction. Insoluble, yeast-derived ENV proteins are being considered as vaccine antigens and the P. pastoris system offers an efficient method of production.

Amino Acid Sequence↗
PubMed2001

Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL-1.

To optimize the secretory expression of recombinant human serum albumin (HSA) under the control of methanol oxidase (MOX) promoter in the methylotrophic yeast Hansenula polymorpha DL-1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5'-untranslated region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational level. With the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus, a single copy of the expression vector integrated into the MOX locus became sufficient for the maximal expression of HSA. Northern blot analysis revealed that the levels of HSA transcript did not increase any further upon increasing copy number. The mox-disrupted (mox Delta) transformant was constructed, in which the genomic MOX gene was transplaced with the HSA expression cassette, to examine the effect of the methanol oxidase-deficient phenotype of the host on HSA expression. The mox Delta transformant showed higher levels of HSA production in shake-flask cultures than the MOX wild-type transformant, especially at low concentrations of methanol and a twofold higher specific HSA production rate in fed-batch fermentation with an abrupt induction mode. The native prepro signal sequence of HSA secreted in H. polymorpha was correctly processed and the mature recombinant protein had a pI value identical to that of the authentic HSA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large-scale production of recombinant albumin.

5' Untranslated Regions↗
PubMed1986

Idiotope-specific T cell clones that recognize syngeneic immunoglobulin fragments in the context of class II molecules.

We have established idiotope (Id)-specific T cell lines and clones derived from at least 4 different BALB/c mice immunized with the light chain (lambda 2(315] of the BALB/c myeloma protein M315 (alpha, lambda 2). Independently derived clones were indistinguishable in that they reacted to V lambda 2(315), one or more of the amino acids corresponding to somatically mutated codons 94, 95 and 96 of the third hypervariable region being essential for expression of the Id. While the Id was efficiently expressed on V lambda 2(315), Fv315 and lambda 2(315) fragments, about a 100-1000-fold higher molar concentration of Fab315 and M315 was needed to induce equivalent responses. Thus, Ig quaternary structure heavily influenced the availability of the Id for T cells. The V lambda 2(315)-specific T cells were Thy-1.2+, L3T4+, Ly-2.2- and I-Ed restricted. Some of the T cell clones produced interleukin 2 (IL2), IL3 and B cell growth and differentiation factors upon activation. In addition, T cells were cytotoxic in long-term assays for Ed beta Ek alpha-, but not Ek beta Ek alpha- transfected L cells in the presence of Id. The cytotoxic effect was the basis for an L cell growth inhibition assay for T cell activation that was at least 10-fold more sensitive than ordinary proliferation assays.

Animals↗
PubMed2005

Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli.

Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein and 5-lipoxygenase-activating protein (FLAP) are among a large number of membrane proteins that are poorly expressed when traditional expression systems and methods are employed. Therefore to efficiently express difficult membrane proteins, molecular biologists will have to develop novel or innovative expression systems. To this end, we have expressed the SARS-CoV M and FLAP proteins in Escherichia coli by utilizing a novel gene fusion expression system that takes advantage of the natural chaperoning properties of the SUMO (small ubiquitin-related modifier) tag. These chaperoning properties facilitate proper protein folding, which enhances the solubility and biological activity of the purified protein. In addition to these advantages, we found that SUMO Protease 1, can cleave the SUMO fusion high specificity to generate native protein. Herein, we demonstrate that the expression of FLAP and SARS-CoV membrane proteins are greatly enhanced by SUMO fusions in E. coli.

5-Lipoxygenase-Activating Proteins↗
PubMed1993

High expression of the hormone binding active extracellular domain (1-294) of rat lutropin receptor in Escherichia coli.

Using the polymerase chain reaction (PCR) technique, the cDNA fragment corresponding to the receptor coding region for residues 1-294 was prepared from rat lutropin receptor (LHR) cDNA and subsequently subcloned into Escherichia coli expression vector pT7-7. This truncated receptor was efficiently expressed in E. coli as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The recombinant protein present in the inclusion bodies was solubilized in 6 M guanidine-HCl and purified in two successive steps of fast performance liquid chromatography (FPLC) using Superose-12 and Mono Q columns. Refolding of the purified recombinant protein was achieved in 1.5 M guanidine-HCl in the presence of an equimolar proportion of cysteine and cystine. The refolded soluble truncated LHR(1-294) had apparent molecular weights of 33 kDa and 140 kDa under reducing and nonreducing conditions, respectively. The multimeric nature of the extracellular domain of the receptor is believed to be due to its self-association by intermolecular disulfide bond formation since the 1-294 amino acid segment has nine cysteine residues and thus has one or possibly more free sulfhydryl groups. The purified truncated receptor had high binding affinity for human choriogonadotropin (hCG) as indicated by ligand blotting on SDS-PAGE and radioligand receptor assays. The amount of hCG required for 50% inhibition of binding of 125I-hCG to the soluble truncated receptor was 2.7 x 10(-10) M (or 12.25 ng). Similarly, the amount of soluble truncated receptor required for 50% inhibition of 125I-hCG binding to the rat ovarian receptor in the radioligand receptor assay was 58 ng.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Intracellular production of a major cytomegalovirus antigenic protein in the methylotrophic yeast Pichia pastoris.

We have previously shown that single or multiple epitopes of the major human cytomegalovirus (HCMV) antigens, produced as fusion proteins in prokaryotes can be valuable diagnostic material in the serology of HCMV infection. In this work we moved to a eukaryotic system, to produce one of the most immunogenic HCMV antigens, ppUL44 (also called pp52 due to its apparent molecular size on acrylamide gels), as a non-fusion protein, in an attempt to eliminate some non-specific reactivity of human sera with bacterial carrier proteins. We expressed the DNA encoding ppUL44 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. Good levels of intracellular, soluble pp52 were produced. We observed an indistinguishable pattern of the yeast pp52 from the viral native protein in immunoblotting and a good reactivity with human sera.

Antibodies, Viral↗
PubMed2005

Immunogenicity and cytoadherence of recombinant heparin binding haemagglutinin (HBHA) of Mycobacterium avium subsp. paratuberculosis: functional promiscuity or a role in virulence?

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's Disease, a chronic granulomatous enteritis of ruminants. Recently, an association of MAP bacilli with Crohn's disease in humans has been proposed. Due to genetic similarities and serological cross-reactivity of the M. avium complex with other mycobacteria, functional analysis of species-specific proteins may allow new insights into the pathogenesis of mycobacterial diseases. We report production and molecular characterization of the recombinant HBHA from the MAP complex bacilli. The HBHA was expressed in Escherichia coli and Mycobacterium smegmatis using efficient expression vector systems. The recombinant HBHA was found to be immunogenic and therefore induced antibody responses in cattle against the MAP bacilli with a possible cross reactivity with M. bovis infection. The MAP complex HBHA was thus found to be a target of the host humoral responses in Johne's disease. The recombinant HBHA protein was also found to be adherent to the Caco2 cell lines in-vitro, a significant observation to understand possible virulence mechanisms. Since M. tuberculosis HBHA was earlier shown to be involved in dissemination of the tubercle bacilli, the immunogenicity and cytoadherent nature of this MAP protein possibly suggests functional promiscuity.

Amino Acid Sequence↗
PubMed1997

Development and characterization of a binary gene expression system based on bacteriophage T7 components in adenovirus vectors.

To explore the utility of the bacteriophage T7 binary system in adenovirus (Ad) vectors we constructed three Ad5-based vectors containing the T7 RNA polymerase (T7pol) gene in either early region 1 (E1) or E3. The recombinant Ad vectors were either deficient (AdT7pol1, AdT7pol2) or competent (AdT7pol3) for replication in human cells other than Ad5 transformed (293) cells. To test the ability of the T7 polymerase produced by these vectors to drive gene expression, a reporter vector was constructed with an E1 substitution comprising the bacterial beta-galactosidase (betaGal) (lacZ) gene under the control of the T7 gene 10 promoter (T7pro) and linked to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) (AdBHG10T7betaGal). Coinfections were performed with the various AdT7pol vectors and the reporter vector, and expression was analysed in three different human cell lines: 293, A549 and MRC-5. Depending on the AdT7pol vector used, different levels of expression were obtained from the reporter gene. In 293 cells, expression was detected following infection at very low multiplicities of infection (moi) with all of the T7pol vectors when coinfected with the reporter vector AdBHG10T7betaGal. In A549 and MRC-5 cells very little expression was detected using AdT7pol1 or pol2 and efficient expression was only obtained when relatively high moi values of the replication-competent vector were used in the coinfections. We also constructed a single vector containing both elements of the T7 system (T7pol in E3 and T7 promoter driving expression of the chloramphenicol acetyl transferase (cat) gene in E1). This vector proved difficult to rescue but was stable once isolated. Finally, experiments performed to evaluate the 'leakiness' of the Ad-T7 system detected very little expression from the T7pro in the absence of T7 polymerase suggesting this system may be useful for the cloning and expression of genes encoding cytotoxic proteins.

Adenoviridae↗
PubMed2005

Expression of porcine acid-labile subunit (pALS) of the 150-kilodalton ternary insulin-like growth factor complex and initial characterization of recombinant pALS protein.

Acid-labile subunit (ALS) is a component of the 150-kDa insulin-like growth factor-binding protein-3 (IGFBP-3) complex, which, by sequestering the majority of IGFs-I and -II and thereby prolonging the half-life of them in plasma, serves as a circulating reservoir of IGFs in mammalian species. A pGEX-2T plasmid and a baculovirus expression constructs harboring a coding sequence for glutathione-S-transferase (GST)-porcine ALS (pALS) fusion protein were expressed in BL21(DE3) E. coli and Sf9 insect cells, respectively. The expressed protein was purified by glutathione or Ni-NTN affinity chromatography, followed by cleavage of the fusion protein using Factor Xa. In addition, pALS and hIGFBP-3 were also produced in small amounts in the Xenopus oocyte expression system which does not require any purification procedure. A 65-kDa pALS polypeptide was obtained following the prokaryotic expression and the enzymatic digestion, but biochemical characterization of this polypeptide was precluded because of an extremely low expression efficiency. The baculovirus as well as Xenopus-expressed pALS exhibited the expected molecular mass of 85 kDa which was reduced into 75 and 65 kDa following deglycosylation of Asn-linked carbohydrates by Endo-F glycosidase, indicating that the expressed pALS was properly glycosylated. Moreover, irrespective of the source of pALS, the recombinant pALS and hIGFBP-3 formed a 130-kDa binary complex which could be immunoprecipitated by anti-hIGFBP-3 antibodies. Collectively, results indicate that an authentic pALS protein can be produced by the current expression systems.

Animals↗
PubMed2002

Expression of p14ARF overcomes tumor resistance to p53.

Tumors without p53 mutation are often resistant to p53 gene therapy. We examined the mechanism using p53-resistant A549 cells and p53-sensitive H1299 cells. We found that p53 delivered by adenovirus is poorly expressed in A549 (ARF-null) cells but efficiently expressed in H1299 cells (ARF-positive). Strong p53 expression and apoptosis can be achieved in A549 cells using a p53 mutant resistant to degradation by MDM2 or by coexpression of ARF. The results suggest that enhanced MDM2 activity attributable to loss of ARF contributes to p53 resistance. Surprisingly, tumor cell lines with MDM2 gene amplification are still deficient for ARF expression, suggesting that MDM2 amplification does not substitute for ARF inactivation during tumor development.

Apoptosis↗