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Results for “Recombinant expression”

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PubMed2004

Molecular definition of predictive indicators of stable protein expression in recombinant NS0 myeloma cells.

We have generated a molecular description of the loci at which stability/instability of expression of a monoclonal antibody (MAb) (anti-CD38) occurs within the GS-NS0 expression system. Critically, these data show that, in the absence of changes to copy number for the recombinant gene sequences, all cell lines examined exhibit a progressive loss (instability) in expression of mRNA during prolonged culture. However, not all cell lines express instability at the level of MAb protein production. The molecular distinction between stable and unstable production at the protein level is a reflection of the cellular amount of recombinant mRNA encoding MAb. Our data indicate a threshold level, a putative saturation point for utilisation of mRNA in translational/secretory events, that defines stability or instability of protein production. Above this level of recombinant mRNA expression, cell lines are stable, whereas below this level cell lines will show instability of protein production. Our studies indicate that absolute levels of expression of recombinant mRNA encoding for MAb in the GS-NS0 expression system offer a potential predictive indicator for the selection of stable cell lines for scale-up. These studies identify molecular facets of host cell biology of generic interest for gene regulation and expression and define techniques and approaches for enhancement of recombinant protein expression and process development.

ADP-ribosyl Cyclase↗

PubMed2005

The use of a recombinant baculovirus expressing a chitinase from the hard tick Haemaphysalis longicornis and its potential application as a bioacaricide for tick control.

Baculoviruses are specific insect pathogens used as selective biological insecticides on lepidopteran insects. We have tested a recombinant baculovirus expressing a chitinase gene for its efficacy as a tick bioacaricide. The recombinant Autographa californica multiple nuclear polyhedrosis virus expressing a chitinase enzyme (AcMNPV-CHT1) from the hard tick, Haemaphysalis longicornis, was constructed and found to have a novel bioacaricidal effect against ticks. The recombinant baculovirus was used to express the chitinase enzyme in Spodoptera frugiperda (Sf9) insect cells. Topical application of the supernatant harvested from the insect cell culture was found to cause mortality in nymphal ticks of H. longicornis. High temperature (>30 degrees C) and infrared radiation affected the chitinase enzyme activity and recombinant baculovirus infectivity by reducing the speed of tick killing by 60%. A mixture of recombinant virus and chitinase was found to kill ticks faster (p < 0.01) than pure chitinase and recombinant virus alone. Thus, the recombinant virus showed a synergistic effect with the foreign chitinase gene. In order to reduce the excessive use and cost of acaricides, it was found that a mixture of recombinant virus and flumethrin could halve the dose of the chemical acaricide used. These findings are important for the safe use of the recombinant virus expressing chitinase as a bioacaricide against ticks.

Animals↗

PubMed1997

Agonist-independent effect of an allosteric enhancer of the A1 adenosine receptor in CHO cells stably expressing the recombinant human A1 receptor.

The allosteric enhancer PD 81,723, a 2-amino-3-benzoylthiophene derivative, has been shown to potentiate agonist binding to A1 adenosine receptors (A1AdoRs) and to enhance the functional effects of adenosine and adenosine analogs. The objective of this study was to determine whether the apparent agonist-independent effect of PD 81,723 observed in CHO cells stably expressing the recombinant human A1AdoR was due to the potentiation of the action of endogenous adenosine, to the presence of constitutive receptor activity and/or to the binding of PD 81,723 to the agonist binding site of the A1AdoR. The allosteric enhancer PD 81,723, the A1AdoR agonist (R)-N6-(2-phenylisopropyl)adenosine and adenosine all significantly inhibited forskolin-stimulated cAMP accumulation in intact cells and increased [35S]-5'-(gamma-thio)triphosphate binding to cell membranes. The effects of adenosine on cAMP formation and [35S]-5'-(gamma-thio)triphosphate binding were attenuated by adenosine deaminase, but the effects of PD 81,723 were not. In the presence of ADA, the A1AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine increased forskolin-stimulated cAMP accumulation in cells expressing the recombinant human A1AdoR but not in nontransfected CHO cells. In binding experiments, the agonist (R)-N6-(2-phenylisopropyl)adenosine, but not PD 81,723, significantly displaced the specific binding of the A1AdoR agonist [3H]-N6-cyclohexyladenosine and the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine. The results of this study demonstrate that in CHO cells stably expressing the recombinant human A1AdoR, the agonist-independent effect of PD 81,723 is not due to potentiation of the action of endogenous adenosine or mediated by the binding of the allosteric enhancer to the agonist binding site of the recombinant human A1AdoR. It is possible that these effects are due to potentiation of constitutive receptor activity by PD 81,723.

Adenosine↗

PubMed2001

[Transfer and Expression of Recombinant Human Thrombopoietin Gene in COS-7 Cells and Mice In Vivo]

In order to investigate the expression of recombinant TPO gene in COS-7 cells and in vivo of the mouse model, eukaryotic expressing plasmid pcd2/TPO with human TPO cDNA was constructed with DNA recombinant techniques. The plasmid pcd2/TPO was transiently transfected into the COS-7 cells by means of lipofection, the naked pcd2/TPO plasmid was injected into the skeletal muscle of mice with electric pulses. RT-PCR and ELISA methods were used to detect the TPO expression of the transfected COS-7 cells, both showed high level expression. The MTT test showed the expressed TPO had proliferative activity to TPO-dependent cell line. High efficiency of gene transfer in transgenic mice was also observed by RT-PCR and immunohistochemical methods. The serum TPO level [(1 185 +/- 264) ng/L] in transgenic mice was quite different compared with the normal mice [(250 +/- 76) ng/L]. All these results provided solid foundations for the research of TPO gene therapy in the future.

Journal Article↗

PubMed1997

Expression of cytochrome P450 3A7 in Escherichia coli: effects of 5' modification and catalytic characterization of recombinant enzyme expressed in bicistronic format with NADPH-cytochrome P450 reductase.

Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation. We report the development of bacterial expression systems for P450 3A7. Maximal yields (up to 50 nmol P450/liter culture) were obtained with a construct in which the 5'-terminus of the 3A7 cDNA was modified to include the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and to incorporate several downstream amino acid substitutions derived from the P450 3A5 sequence. This sequence also appeared optimal for expression of P450 3A4 and 3A5. Recombinant P450 3A7 was partially purified using ion-exchange and hydroxylapatite chromatography and reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, and lipids. Activity comparable to that of P450 3A4 was demonstrated toward a number of procarcinogens. An alternative approach was used to further characterize recombinant 3A7 due to low yields of recombinant protein in the expression and poor recovery in the purification. P450 3A7 was subcloned into a bicistronic vector containing human NADPH-cytochrome P450 reductase and expressed in bacteria. Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation).

Amino Acid Sequence↗

PubMed1994

Renaturation and purification of human tissue factor pathway inhibitor expressed in recombinant E. coli.

Tissue factor pathway inhibitor is an inhibitor of the extrinsic coagulation pathway. Evaluation of the pharmacological effects of tissue factor pathway inhibitor in animal models has been limited by the high cost and low availability of mammalian tissue culture produced protein. In order to circumvent this obstacle, a 277-amino-acid nonglycosylated tissue factor pathway inhibitor variant possessing an N-terminal alanine was expressed in recombinant E. coli using the tac promoter expression system. High-level expression in recombinant E. coli resulted in the accumulation of ala-tissue factor pathway inhibitor in inclusion bodies. Active protein was produced by solubilization of the inclusion bodies in 8 M urea, purification of the full-length molecule by cation exchange chromatography, and renaturation in 6 M urea. Fractionation of crude refold mixtures using cation exchange chromatography yielded a purified nonglycosylated tissue factor pathway inhibitor possessing in vitro prothrombin time activity comparable to inhibitor purified from mammalian cell lines.

Amino Acid Sequence↗

PubMed2003

Reduced experimental autoimmune encephalomyelitis after intranasal and oral administration of recombinant lactobacilli expressing myelin antigens.

Oral administration of autoantigens is a safe and convenient way to induce peripheral T-cell tolerance in autoimmune diseases like multiple sclerosis (MS). To increase the efficacy of oral tolerance induction and obviate the need for large-scale purification of human myelin proteins, we use genetically modified lactobacilli expressing myelin antigens. A panel of recombinant lactobacilli was constructed producing myelin proteins and peptides, including human and guinea pig myelin basic protein (MBP) and proteolipid protein peptide 139-151 (PLP(139-151)). In this study we examined whether these Lactobacillus recombinants are able to induce oral and intranasal tolerance in an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Lewis rats received soluble cell extracts of Lactobacillus transformants intranasally three times prior to induction of EAE. For the induction of oral tolerance, rats were fed live transformed lactobacilli for 20 days. Ten days after the first oral administration EAE was induced. Intranasal administration of extracts containing guinea pig MBP (gpMBP) or MBP(72-85) significantly inhibited EAE in Lewis rats. Extracts of control transformants did not reduce EAE. Live lactobacilli expressing guinea pig MBP(72-85) fused to the marker enzyme beta-glucuronidase (beta-gluc) were also able to significantly reduce disease when administered orally. In conclusion, these experiments provide proof of principle that lactobacilli expressing myelin antigens reduce EAE after mucosal (intranasal and oral) administration. This novel method of mucosal tolerance induction by mucosal administration of recombinant lactobacilli expressing relevant autoantigens could find applications in autoimmune disease in general, such as multiple sclerosis, rheumatoid arthritis and uveitis.

Administration, Intranasal↗

PubMed2000

[Construction and expression of recombinant adeno-associated HIV -1 virus].

OBJECTIVE: Plasmid of AAV vector with HIV-1 gag and gp 120 were constructed respectively. METHODS: The recombinant AAV-HIV gag and gp120 viruses were obtained by co transfection of AAV-HIV plasmid and Adenovirus 5 in 293 cells. RESULTS: The recombinant AAV-HIV virus expressed well and the titer of the recombinant viruses were between 10(4)-10(5). CONCLUSIONS: The recombinant AAV-HIV virus can be used to infect the target cells for detection of cellular immunity and for vaccine studies.

Adenoviruses, Human↗

PubMed1991

Expression of the outer capsid protein VP5 of two bluetongue viruses, and synthesis of chimeric double-shelled virus-like particles using combinations of recombinant baculoviruses.

We have previously reported the assembly of virus-like particles (VLPs), consisting of the four major structural proteins of bluetongue virus (BTV), in Spodoptera frugiperda cells coinfected with recombinant baculoviruses (French et al. (1990). J. Virol. 64, 5695-5700). In this paper we report further studies using this system to assemble heterologous VLPs containing the outer capsid proteins (VP2 and VP5) of a range of different BTV serotypes. S. frugiperda cells were coinfected with three recombinant baculoviruses; a dual recombinant expressing VP3 and VP7 (of BTV-17 and -10, respectively) in combination with a single recombinant expressing VP2 of BTV-1, -2, -10, -11, 13, or -17 and an additional single recombinant expressing VP5 of BTV-2, BTV-10, or BTV-13. The resultant VLPs were purified and analyzed by electronmicroscopy, Western immunoblotting, and hemagglutination assays to determine whether double-shelled VLPs had been assembled. In the course of these experiments the VP2 proteins of all six available serotypes were successfully incorporated into VLPs. Particles from two different combinations of chimeric VLPs (having VP2 derived from BTV-1 or that of BTV-17) were used to raise antisera in guinea pigs. Both of these sera showed high neutralizing antibody titers against live BTV, indicating that heterologous VLPs may have potential for use in anti-BTV vaccines.

Animals↗

PubMed1992

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.

Amino Acid Sequence↗

PubMed2003

Expression of recombinant human ICOS and in vitro characterization of its bioactivity on B lymphocytes.

Inducible costimulator (ICOS) is a novel costimulatory molecule expressed in activated T cell and has critical regulation effect on special immune response. In this study, the cDNA encoding human ICOS was cloned from activated tonsil cells via RT-PCR, and was expressed in E. coli on pET28 expression vector. The recombinant ICOS protein expressed from E. coli showed a molecular weight of 14 kD on SDS-polyacrylamide gel electrophoresis and was further confirmed by Western blot. In presence of IL-10, the purified rhICOS significantly increased in vitro B cell growth stimulated by pokeweed mitogen (PWM), and enhanced the secretion of IgG from B cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2006

Functional expression of recombinant human stefin A in mammalian and bacterial cells.

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-1 monkey kidney cells utilizing pET and recombinant vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and Western blot analysis utilizing a polyclonal antibody against rat cystatin alpha. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A ( approximately 10kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BS-C-1 cells, was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells, on the other hand, can provide a significant research tool to study the functional roles of stefin A in mammalian systems such as regulation of cysteine proteases.

Amino Acid Sequence↗

PubMed1990

Vaccination against tumor cells expressing breast cancer epithelial tumor antigen.

Ninety-one percent of breast tumors aberrantly express an epithelial tumor antigen (ETA) identified by monoclonal antibody H23. Vaccinia virus recombinants expressing tumor antigens have considerable promise in the active immunotherapy of cancer, and we have evaluated the potential of vaccinia recombinants expressing the secreted (S) and cell-associated (transmembrane, T) forms of H23 ETA to elicit immunity to tumor cells expressing ETA. Tumorigenic ras-transformed Fischer rat fibroblast lines FR-S and FR-T, expressing the S or T form of H23 ETA, respectively, were constructed for use in challenge experiments. Expression of H23 ETA in these lines was confirmed by Western blotting and immunofluorescence. When challenged by subcutaneous seeding of tumor cells, 97% (FR-S) and 91% (FR-T) of syngeneic Fischer rats rapidly developed tumors that failed to regress. Vaccination with recombinant vaccinia virus expressing ETA-T prior to challenge prevented tumor development in 82% of animals seeded with FR-T cells but in only 61% of animals seeded with FR-S. The vaccinia recombinant expressing the S form was a less effective immunogen, and vaccination protected only 29-30% of animals from developing tumors upon challenge with either FR-S or -T cells. The increased immunogenicity of the recombinant expressing ETA-T was reflected in elevated levels of ETA-reactive antibody in vaccinated animals, confirming that secreted antigens expressed from vaccinia virus are less effective immunogens than their membrane-associated counterparts.

Animals↗

PubMed1995

Mitomycin C stimulates thermally induced recombinant gene expression in Escherichia coli MC strains.

The effects of mitomycin C on C1857-controlled recombinant gene expression have been explored in E. coli cultures when the drug was added simultaneously to the thermal induction. A significantly improved yield of homologous, heterologous and chimeric fusion proteins was observed in E. coli MC1061 and GE864 (a MC4100 derivative) thermoinduced cells. This feature was not detected in other E. coli strains and does not involve a gene dosage mechanism but a strain-dependent stimulation of gene expression unrelated to the RecA protease activity.

DNA, Bacterial↗

PubMed2005

Rainbow tags: a visual tag system for recombinant protein expression and purification.

Visualization systems for tracking proteins are standard experimental tools in most areas of biological research apart from protein purification. Here, we have sought to plug this gap by producing red and yellow visual tags using the heme-binding domain of mosquito cytochrome b5 and the flavin mononucleotide (FMN)-binding domain of human P450 reductase. Tests with colorless glutathione-S-transferase (GST) show them to be simple and effective tools for visually identifying correctly folded protein and tracking protein molecules through protein expression and purification. Furthermore, the characteristic absorbance signatures of the colored tags can be used to quantify protein concentrations directly, which allows purification to be linked to colorimetric detection. This technology, which we call Rainbow Tagging, facilitates expression and downstream processing of recombinant proteins, paving the way for the development of automated high-throughput protein expression systems.

Animals↗

PubMed1996

Mutant forms of the F protein of human respiratory syncytial (RS) virus induce a cytotoxic T lymphocyte response but not a neutralizing antibody response and only transient resistance to RS virus infection.

Vaccinia virus (vv) recombinants expressing either wild-type (VA-F) or mutant forms (VA-FT, VA-FR47, VA-FS1 to VA-FS6) of the fusion (F) protein of respiratory syncytial (RS) virus were examined for their ability to elicit antibody, cytotoxic T lymphocytes (CTL) and protection against RS virus infection in BALB/c mice. Cells infected with the VA-F and VA-FT recombinants expressed the F protein on their surface and mice vaccinated with these recombinants developed RS virus neutralizing antibodies. The VA-FR47 recombinant expressed a mutant form of the F protein (with six amino acid changes from the wild-type) in which both proteolytic processing of the F0 precursor and its transport to the cell surface were inhibited. These mutants induced transient protection against RS virus infection although they did not induce RS virus neutralizing antibodies, or antibodies detectable by ELISA. All the vv recombinants were able to induce an RS virus-specific, MHC class I restricted CTL response. Vaccination of mice with a second set of vv recombinants expressing mutant forms of the F protein showed that the replacement Phe to Ser at amino acid 237 either alone or in combination with others abolished the neutralizing antibody response but did not affect priming of CTLs. These results demonstrate that long-term protection against RS virus infection in mice vaccinated with recombinant vv expressing the F protein is more dependent upon the induction of an antibody rather than a CTL response.

Animals↗

PubMed1993

Analysis of bovine herpesvirus 1 glycoprotein gIV truncations and deletions expressed by recombinant vaccinia viruses.

Glycoprotein gIV is an envelope component of bovine herpesvirus type 1 and appears to be involved in attachment, penetration, and cell fusion. Four antigenic domains which include both continuous and discontinuous epitopes have been previously defined by competition binding assays using gIV-specific monoclonal antibodies (MAbs). Here we describe the construction of C-terminal truncations and internal deletions in the gIV-encoding gene and analyses of the effects of these mutations on the synthesis, processing, transport, and antigenicity of glycoprotein gIV as expressed by recombinant vaccinia viruses. Wild-type gIV expressed by recombinant vaccinia virus STgIV was indistinguishable from authentic gIV produced in bovine herpesvirus 1-infected cells with respect to molecular weight, processing, transport, and antigenicity. Analysis of the mutant proteins showed that the binding sites for MAbs 9D6 and 3D9S, which recognize linear epitopes, lie between amino acids 164 and 216 and amino acids 320 and 355, respectively. Discontinuous epitopes recognized by MAbs 3E7, 4C1, 2C8, and 3C1 were located between amino acids 19 and 320, whereas amino acids 320 to 355 were critical for binding of MAb 136. All mutant proteins containing amino acids 245 to 320 were processed, possess endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides, and were transported to the cell surface or secreted into the medium. In contrast, mutant proteins missing amino acids 245 to 320 were retained in the rough endoplasmic reticulum. These findings suggest that residues 245 to 320 are important for proper processing and transport of gIV to the cell surface.

Animals↗

PubMed1994

The effect of N-linked glycosylation on activity of the Na(+)- and Cl(-)-dependent serotonin transporter expressed using recombinant baculovirus in insect cells.

The rat Na(+)- and Cl(-)-dependent serotonin transporter was expressed in Sf9 insect cells using the baculovirus system. Expression of the serotonin transporter caused the Sf9 cells to accumulate [3H]serotonin (Km 78 nM) and to bind the specific transport inhibitor [125I]RT155 (2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane) (Kd 0.22 nM). Ligand binding assays on isolated membranes showed 500,000 copies of the serotonin transporter/cell (9 pmol/mg of membrane protein). Immunoreactive bands of apparent M(r) 54,000 (unglycosylated) and 60,000 (glycosylated) were observed in Western blots of membrane proteins from infected cells. The 54-kDa band was significantly smaller than the expected M(r) of 72,500 predicted from the cDNA sequence. The 54-kDa band was shown to represent the intact serotonin transporter by expressing a recombinant serotonin transporter that contained c-Myc and FLAG epitope tags engineered at the N and C termini, respectively. Both tags were present on a membrane protein that migrated slightly slower than the previously observed 54-kDa band, consistent with the extra mass added by the tags. The tags did not affect the Kd for [125I]RT155 binding. The effect of N-linked glycosylation on ligand binding and the level of expression were studied. The expression of the serotonin transporter in tunicamycin-treated Sf9 cells resulted in low levels of ligand binding activity (0.2 pmol/mg) but unchanged Kd. Similarly, mutated serotonin transporters that contained reduced numbers of N-linked glycosylation sites had unchanged Kd for [125I]RT155 binding whether there were 2, 1, or 0 N-linked glycosylation sites present on the serotonin transporter. In contrast, Bmax was dramatically reduced; levels of expression of the unglycosylated serotonin transporter (0.4 pmol/mg) were 20-fold lower compared with levels of the fully glycosylated serotonin transporter. The Km for [3H]serotonin uptake was also unchanged. These data indicate that glycosylation is required for optimal stability of the serotonin transporter in the membrane but not for serotonin transport or ligand binding per se.

Animals↗