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Results for “Recombinant expression”

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PubMed2001

Recombinant expression and purification of human androgen receptor in a baculovirus system.

A full-length human androgen receptor (hAR) cDNA was used to produce recombinant baculovirus. Spodoptera frugiperda (Sf9) cells infected with this virus expressed protein with an N-terminal hexahistidine tag (His(6)-hAR) in soluble and insoluble forms. The soluble cytosolic His(6)-hAR demonstrated similar association and dissociation half-times for mibolerone, similar binding affinity for mibolerone, and similar steroid specificity as bona fide AR. Under native conditions, the soluble cytosolic His(6)-hAR was purified to apparent homogeneity in the presence of dihydrotestosterone, using metal ion affinity chromatography. The insoluble pellet fraction was solubilized with strong denaturant 6 M guanidine HCl, and His(6)-hAR was purified from it in the presence of 6 M guanidine HCl. Both the solubilized crude pellet fraction and the solubilized/purified His(6)-hAR could be renatured to bind mibolerone. The baculovirus system will therefore provide an efficient means for producing hAR for ligand-binding assays, as well as purifying hAR for detailed molecular analyses.

Animals↗

PubMed1997

Metabolism of catecholamines by catechol-O-methyltransferase in cells expressing recombinant catecholamine transporters.

To determine if catechol-O-methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3H-substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3H-catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Km of transport from 0.61 microM to 0.34 microM without significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.

Carrier Proteins↗

PubMed1988

Vaccinia virus recombinants expressing an 11-kilodalton beta-galactosidase fusion protein incorporate active beta-galactosidase in virus particles.

Recombinant plasmids in which vaccinia virus transcriptional regulatory sequences were fused to the Escherichia coli lacZ gene were constructed for insertion of the lacZ gene into the vaccinia virus genome. beta-Galactosidase (beta-gal) was found in some purified recombinant vaccinia virions. By enzyme activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and microscopic techniques, the evidence suggested that beta-gal accounted for 5% of the total protein in the virion. These recombinant viruses were constructed so that a portion of the coding sequences of a late vaccinia virus structural polypeptide was fused to the amino terminus of beta-gal to produce the fusion protein. Removal of the coding sequences resulted in the complete loss of beta-gal activity. This demonstrated that a vaccinia virus DNA segment from a late structural gene is responsible for the incorporation of beta-gal into the virion.

Base Sequence↗

PubMed1989

Immune response and protection of cattle and pigs generated by a vaccinia virus recombinant expressing the F protein of rinderpest virus.

The immune response of cattle and pigs to a vaccinia recombinant virus containing the fusion (F) protein gene of rinderpest virus was examined. Half the cattle and all the pigs gave humoral response to primary vaccination and all the cattle gave an anamnestic response to a second vaccination 28 days after the primary vaccination. All the cattle after a single or secondary vaccination were completely protected clinically after exposure to a lethal dose of the Saudi 1/81 strain of virus. Prior vaccination with another TK- vaccinia recombinant (VVCAT) suppressed, but did not abrogate, the immune response to the rinderpest F recombinant. The pigs gave a humoral immune response in the absence of any local reaction at the site of vaccination.

Animals↗

PubMed1988

Epstein-Barr virus (EBV) infection of murine L cells expressing recombinant human EBV/C3d receptor.

The normal host range of Epstein-Barr virus (EBV) is limited to primate B lymphocytes and certain epithelial cells that express the C3d/EBV receptor [complement receptor 2 (CR2, CD21)]. In the present study, expansion of the tissue tropism of EBV has been accomplished by stably transfecting the murine fibroblast L cell line with pMT.CR2. neo.1, a eukaryotic expression vector promoting the transcription of a complementary DNA insert encoding human CR2. High CR2-expressing transfected L cells were selected by fluorescence-activated cell sorting. The recombinant CR2 was shown to have the same molecular weight as wild-type CR2 from Raji cells and to mediate the binding by the transfectants of particles bearing the iC3b and C3d fragments of the third component of complement. All CR2-expressing L cells, but not nontransfected controls, also bound EBV, as assessed by indirect immunofluorescence. After a 60-hr culture, approximately 0.5% of the CR2-expressing cells preincubated with EBV demonstrated immunofluorescent staining of EBV nuclear antigen with serum from a patient with nasopharyngeal carcinoma. No fluorescent staining of cells was seen with monoclonal antibodies to the early antigen complex or to gp350/220, indicating that the infection was predominantly latent. Infected cells cultured for up to 4 weeks remained EBV nuclear antigen-positive. The capacity of recombinant human CR2 to confer on murine L cells susceptibility to stable latent infection by EBV indicates that this receptor is a primary determinant of the tissue tropism of EBV and may facilitate studies of cell-specific factors that regulate the viral growth cycle.

Animals↗

PubMed1997

Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants.

A potential role for cAMP in regulating the differentiation of myoblasts has led us to examine the components of the cAMP signaling system, including the type IV, cAMP-specific phosphodiesterases. The full coding sequence of the phosphodiesterase PDE4D1 was inserted in the bacterial expression vector pGEX-KG. N- and C-terminal truncations were also placed in the same vector, allowing the expression and purification of glutathione S-transferase (GST)-PDE fusion proteins using glutathione-Sepharose. The purified PDE was active [V(max) = 318 +/- 18 nmol min(-1)(mg of protein)(-1)] and inhibited by RO 20-1724, rolipram, and MIX (IC50 values of 2, 0.4, and 40 microM, respectively). The requirement of PDE4D1 for a divalent cation was also examined. It was able to use Mg2+, Co2+, and Mn2+, but not Zn2+, suggesting that it is not a zinc hydrolase as has been proposed for other PDE types. Deletion of both C- and N-terminal regions affected the apparent native size of the enzyme. The C-terminal region was involved in dimer formation, whereas an N-terminal region was responsible for larger aggregates. Removal of the last 35 amino acids of an N-terminal 80-residue highly conserved region (UCR2) resulted in a 6-fold increase in PDE activity, providing evidence that this part of the molecule acts as an intramolecular inhibitor. The availability of a highly purified, enzymatically active protein in substantial quantities has allowed us to directly examine PDE4D1 for the first time.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1991

Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase.

Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the convenient purification of 15-20 mg enzyme from 11 culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.

Chromatography, Gel↗

PubMed1998

Construction of human insulin gene expression recombinant and its effect on blood glucose of diabetic rats.

OBJECTIVE: To study the effect of human insulin gene modified fibroblasts on blood glucose in diabetic rats. METHODS: An expression vector containing human insulin gene was constructed by recombinant DNA technique and introduced into fibroblast Ltk- cells by lipofectin-mediated DNA transfection. Following G418 screening, the survived cells were selected and enriched. Finally a cell line, PRI-12 was generated for the highest insulin production. Then, these cells were injected into streptozocin (STZ)-induced diabetic rats. RESULTS: Insulin DNA transfected Ltk- cells were able to express insulin at a high level in a long-term culture. Furthermore, these Ltk- transfectants could decrease blood glucose significantly (P < 0.01) and obviously increase the body weight (P < 0.01) when they were injected into STZ-induced diabetic rats. CONCLUSIONS: These results suggest that the cell lines transfected insulin gene could secrete insulin and execute effect on diabetic rats. The study provides support for the view that somatic cell gene therapy offers a potential approach to delivery insulin into diabetes mellitus.

Animals↗

PubMed2004

Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR.

Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (pI 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris. The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC. N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein. NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6. The recombinant form of Ole e 6 expressed in P. pastoris can be employed for structural and biochemical studies.

Chromatography↗

PubMed1993

Recombinant expression of the pufQ gene of Rhodobacter capsulatus.

Genetic studies have shown that the expression of the pufQ gene is required for normal levels of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus. Yet, the exact function of the pufQ gene is unknown, and a pufQ gene product has never been isolated. We describe the recombinant overexpression of pufQ in Escherichia coli, as well as the purification and characterization of its gene product, the 74-amino-acid PufQ protein. Site-directed mutagenesis was used to facilitate the cloning of the pufQ gene into various expression vector systems of E. coli, including pKK223-3, pLcII-FX, and pMal-c. Although high levels of pufQ transcription were evident from constructs of all three vectors, high levels of protein expression were apparent only in the pMal-c system. In vector pMal-c, the recombinant PufQ protein is expressed as a fusion with an amino-terminal maltose-binding domain. After affinity purification on an amylose column, full-length PufQ protein was released from the fusion protein by limited proteolysis with the enzyme factor Xa. The PufQ protein demonstrated a strong tendency to associate with phospholipid vesicles, consistent with the view that it is an integral membrane protein. The PufQ protein was subsequently purified by high-performance liquid chromatography and identified by amino-terminal sequence analysis. A possible role for the PufQ protein in the transport of bacteriochlorophyll biosynthetic intermediates is discussed.

Bacterial Proteins↗

PubMed1991

beta-Galactosidase expressing recombinant pseudorabies virus for light and electron microscopic study of transneuronally labeled CNS neurons.

A beta-galactosidase expression pseudorabies virus (Bartha strain) was constructed, injected into the adrenal gland of rats, and subsequently shown to transneuronally label the CNS autonomic neurons that project to the sympathoadrenal preganglionic neurons. Virally infected neurons were visualized with a one-step histochemical reaction using the Bluo-Gal substrate (halogenated indolyl-beta-D-galactoside) for the localization of beta-galactosidase activity. In some infected neurons, a Golgi-like staining of the primary and sometimes secondary dendrites could be obtained. For electron microscopic studies, the Bluo-Gal substrate produces an electron-dense reaction product that is easily identified at both low and high magnification. This virus may be useful for the study of the cell architecture and synaptic organization of transneuronally labeled neurons of functionally defined neural circuits. These results also demonstrate that it is possible to deliver foreign genes into specific chains of neurons in the mammalian CNS by means of the retrograde transneuronal vial labeling method.

Adrenal Glands↗

PubMed2006

[Construction and expression of recombinant adenovirus expression vector of beta-defensin-2].

OBJECTIVE: To explore the possibility of stable expression of cationic peptide beta-defensin-2 in eukaryocytes with adenovirus vector. METHODS: The rat beta-defensin-2 (rBD2) gene was cloned at the downstream of CMV promoter of the adenoviral shuttle plasmid pShuttle-CMV. Then pShuttle-CMV-rBD2 was transformed into E. coli BJ5183-AD-1, in which recombination occurred between plasmids and pAdEasy-1 to construct pAdEasy-rBD2. After confirmation by endonuclease, linear pAdEasy-rBD2 was transformed into 292 cells to obtain packaged adenoviral expression vector, which was used to infect cos-7 cells and to establish respiratory adenovirus infection model of rat. The in vivo and in vitro expression activity of recombinant adenovirus was detected by Western blot and immunohistochemistry. RESULT: The inserted DNA of pShuttle-CMV-rBD2 consisted of rat beta-defensin-2 gene. The pathological effect of infected cells, electronic microscopic observation and PCR showed that the recombinant adenovirus vector was constructed successfully. The concentration of the adenovirus was 10(9) PFU/ml. The vector expressed rat beta-defensin-2 efficiently in vivo and in vitro. CONCLUSION: The recombinant adenovirus vector can express cationic peptide beta-defensin-2 in eukaryocytes.

Adenoviridae↗

PubMed1997

Recombinant expression of human microsomal epoxide hydrolase protects V79 Chinese hamster cells from styrene oxide- but not from ethylene oxide-induced DNA strand breaks.

Styrene 7,8-oxide and ethylene oxide are widely used genotoxic bulk chemicals, which have been associated with potential carcinogenic hazard for occupationally exposed workers. Both epoxides alkylate DNA preferentially at the N-7 position of guanine and consequently produce single-strand breaks and alkali labile sites in the DNA of exposed cells. In order to study the role of human microsomal epoxide hydrolase (hmEH) in protecting cells against genotoxicity of styrene 7,8-oxide and ethylene oxide, we expressed the cDNA of hmEH in V79 Chinese hamster cells. We obtained a number of cell clones that expressed functionally active epoxide hydrolase. Among these, the clone 92hmEH-V79 revealed an especially high enzymatic mEH activity toward styrene 7,8-oxide (10 nmol converted per mg of protein per min, measured in the 9,000 x g supernatant of the cell homogenate), that was 100 times higher than that determined in mock-transfected cells and within the range of mEH activity in human liver. Styrene 7,8-oxide-induced DNA single-strand breaks/alkali labile sites (dose range 10 microM to 1 mM styrene 7,8-oxide) measured by the alkaline elution technique were significantly lower in the 92hmEH-V79 cells as compared to the mock-transfected cells. The protection against styrene 7,8-oxide genotoxicity in 92hmEH-V79 cells could be abolished by addition of valpromide, a selective inhibitor of microsomal epoxide hydrolase. These results clearly show that the metabolism of styrene 7,8-oxide by hmEH in 92hmEH-V79 cells was responsible for the protection against styrene 7,8-oxide genotoxicity. On the other hand, no protective effect of epoxide hydrolase expression could be observed on ethylene oxide-induced DNA damage with the recombinant cell line over a dose range of 0.5-2.5 mM ethylene oxide. This selectivity of the protective effect on epoxide genotoxicity thus appears to be an important factor that must be taken into account for the prediction of the genotoxic risk of epoxides themselves or compounds that can be metabolically activated to epoxides.

Animals↗

PubMed2006

Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression.

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 x 10(-6) M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-alpha-d-mannopyranoside.

Aspergillus niger↗

PubMed1993

Selection of Marek's disease virus recombinants expressing the Escherichia coli gpt gene.

We developed a positive selection method for recovering Marek's disease virus (MDV) recombinants. The Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt), under the control of the major immediate-early promoter from cytomegalovirus, was inserted into the inverted repeats flanking the unique long (UL) region of a non-pathogenic serotype 2 MDV strain 281MI/1. In a second demonstration of the usefulness of the positive selection system, the gpt gene was inserted into the inverted repeats flanking the unique short (US) region of the turkey herpesvirus (HVT) strain FC126. The targeted insertion site in 281MI/1 was in a previously established nonessential site for virus replication. The targeted insertion site for FC126, at the junction of the UL and US regions, is a nonessential site for in vitro replication of herpes simplex virus. Recombinant viruses were easily selected by incubating the transfected cells in mycophenolic acid (MPA)-containing medium. Purification of recombinants resulted from a series of trypsinization and sonication steps combined with the culturing of virus in MPA-containing medium to inhibit wild-type virus replication. This simple technique for recovering MDV and HVT recombinants should increase the efficiency of identifying nonessential sites and gene function analysis by insertional mutagenesis.

Blotting, Southern↗

PubMed1997

Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.

Amino Acid Sequence↗

PubMed1999

Recombinant expression of late genes agno-2a and agno-2b of avian polyomavirus BFDV.

Budgerigar fledgling disease virus (BFDV) genome contains two times two (two pairs) open reading frames (agnogenes) at the 5' end of the late coding region. Recombinant influenza A viruses were constructed to express the second pair of BFDV agnoproteins, agno-2a and agno-2b, with a fusion of a histidine-tag at their carboxy-termini, respectively. Specific proteins were detected in Western blot analysis using anti histidine-tag monoclonal antibody. By indirect immunofluorescence experiments agno-2a and agno-2b were shown to be located on the surface and in the perinuclear and cytoplasmic areas of infected cells. Comparisons of the expression patterns of BFDV agno-2a and agno-2b with that of simian virus 40 agnoprotein reveal high similarity, suggesting that they might have the same function(s) in polyomavirus infectious cycle.

Amino Acid Sequence↗

PubMed2005

Lactobacillus acidophilus expressing recombinant K99 adhesive fimbriae has an inhibitory effect on adhesion of enterotoxigenic Escherichia coli.

The most common enteric colibacillosis in neonatal and newborns is caused by enterotoxigenic Escherichia coli(ETEC). Colonization of ETEC in the small intestine is associated with adhesions using fimbriae, which is known as a specific adhesion factor and provides highly specific means for anchoring and prerequisite for an infectious agent. In the present study we have engineered Lactobacillus acidophilus to produce recombinant K99 fimbriae, which is used for the colonization to the intestine of pigs. The expression of K99 fimbrial protein was confirmed using SDS-PAGE, immunoblot and agglutination analyses. To evaluate a function of the K99 fimbrial protein, inhibition and competition tests were performed on pre-screened intestinal brush border from pigs. The tests showed that recombinant L. acidophilus, not control L. acidophilus, had a significant inhibitory effect to and competition against K99+ E. coli in a dose dependent manner. In conclusion, we demonstrated that recombinant K99 fimbriae producing L. acidophilus was able to prevent E. coli binding to intestinal brush border.

Animals↗