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Results for “expression efficiency”

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At least 343 records · Page 19Linked to original sources
PubMed1998

Production and purification of recombinant 2'-5' oligoadenylate synthetase and its mutants using the baculovirus system.

Investigation of the structure-function relationship of the 2'-5' oligoadenylate [2-5 (A)] synthetases has been hampered by the lack of an efficient expression system for a recombinant enzyme. Here, we report that the 9-2 isozyme of murine 2-5 (A) synthetase can be efficiently expressed in insect cells using the baculovirus system. The recombinant protein was purified to apparent homogeneity, and its enzymatic activity was characterized. It had a high specific activity, required double-stranded RNA as a cofactor, and synthesized dimers to hexamers of 2-5 (A). The utility of our expression system was demonstrated by studying the properties of two previously reported mutant proteins. Both of these mutants, when produced in bacteria, are enzymatically inactive, although similarly produced wild-type protein is active. Unexpectedly, when expressed in insect cells, both mutant proteins were enzymatically as active as the wild-type protein. These results suggest that in the eukaryotic expression system described here, the mutant proteins can undergo appropriate modifications or folding that is required for attaining an enzymatically active conformation.

2',5'-Oligoadenylate Synthetase↗
PubMed2002

Priming polyvalent immunity by DNA vaccines expressing chimeric antigens with a stress protein-capturing, viral J-domain.

The N-terminal domain of large tumor antigens (T-Ag) of polyomaviruses forms a DnaJ-like structure with a conserved J domain that associates with constitutively expressed stress protein heat shock protein (hsp)73. Mutant (but not wild-type) SV40 T-Ag show stable, ATP-dependent binding to the stress protein hsp73 when expressed in cells from different vertebrate tissues. Intracellular T/hsp73 complexes accumulate to high steady-state levels. From this observation, we designed a vector system that supports stable expression of a large variety of hsp73-capturing, chimeric antigens containing an N-terminal, T-Ag-derived domain, and different C-terminal antigenic domains from unrelated antigens. Most antigenic domains tested could be stably expressed only in eukaryotic cells as fusion protein/hsp73 complexes. The N-terminal 77 residues representing the J domain of T-Ag were required for stable hsp73 binding and efficient expression of chimeric antigens. Hsp73-bound chimeric antigens expressed by DNA vaccines showed strikingly enhanced immunogenicity evident in humoral (antibody) and cellular cytolytic T lymphocytes (CTL) responses. The described system supports efficient expression of chimeric, polyvalent antigens and their codelivery with hsp73 as a "natural adjuvant" for enhanced immunogenicity for T and B cells.

Amino Acid Sequence↗
PubMed1990

Recombinant gene expression in human umbilical vein endothelial cells transduced by retroviral vectors.

Endothelial cells are attractive targets for gene transfer because of their immediate contact with the bloodstream, and, therefore, they might serve as vehicles for therapeutic drug delivery. Recently, we and others reported that endothelial cells of animal origin efficiently express both secretory and nonsecretory recombinant proteins. We now show that human endothelial cells are also capable of expressing a recombinant gene following transduction with retroviral vectors. Human umbilical vein endothelial cells were transduced with either the N2 or the SAX vector. Following selection with G418, cells transduced by both vectors were found to express neophosphotransferase activity, the product of the neomycin resistance gene. The fact that a recombinant gene can be readily inserted and efficiently expressed into human endothelial cells suggests that these cells may be able to serve a role in human gene therapy.

Anti-Bacterial Agents↗
PubMed2002

Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines.

Mouse embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the developmental capacity to generate all cell types of the body. Combined with efficient genetic manipulation and in vitro differentiation procedures, ES cells are a useful system for the molecular analysis of developmental pathways. We analyzed and compared the transcriptional activities of a cellular polypeptide chain elongation factor 1 alpha (EF), a cellular-virus hybrid (cytomegalo-virus [CMV] immediate early enhancer fused to chicken beta-actin [CBA]), and a viral CMV promoter system in two ES cell lines. When transiently transfected, the EF and CBA promoters robustly drove reporter gene expression, while the CMV promoter was inactive. We also demonstrated that the EF and CBA promoters effectively drove gene expression in different stages of cell development: naïve ES cells, embryoid bodies (EBs), and neuronal precursor cells. In contrast, the CMV promoter did not have transcriptional activity in either ES cells or EB but had significant activity once ES cells differentiated into neuronal precursors. Our data show that individual promoters have different abilities to express reporter gene expression in the ES and other cell types tested.

Actins↗
PubMed1996

Induction of antibodies against structural proteins of hepatitis C virus in mice using recombinant adenovirus.

Replication-deficient recombinant adenoviruses expressing structural proteins of hepatitis C virus (HCV) were constructed. Each recombinant lacks adenoviral E1A and E3 genes and bears expression units for HCV structural proteins. The expression units contain HCV cDNAs coding for either the protein or core, one of two envelopes (E1 and E2) or all of these structural proteins (core, E1 and E2) under the control of the SR alpha promoter. In HeLa or HepG2 cells, the recombinants can express efficiently HCV genes after infection without replication of the recombinants. We detected 22-kDa core, 35-kDa E1 and 58-kDa E2 proteins of HCV in these cells. The recombinant expressing all three HCV structural proteins was inoculated into mice. Antibodies to each of the three HCV proteins were detected in all of the ten mice tested. The results indicate that the recombinant adenoviruses efficiently express HCV genes and induce specific antibody against the expressed HCV proteins in animals.

Adenoviridae↗
PubMed1998

Promoter analysis of the Drosophila genes encoding TFIIB and TATA box-binding protein.

We have analyzed the 5'-flanking regions of the Drosophila genes encoding the TFIIB and TATA box-binding proteins (TBP) and mapped the regulatory regions required for their efficient expression. We found that the 500 bp long region (-439 to +60) and the 401 bp long region (-261 to +138) are required for the efficient expression of TFIIB and TBP genes, respectively. In the TFIIB promoter region, the upstream DNA between -439 and -280 and the downstream DNA between +8 and +60 are necessary for the stimulation of promoter activity. The upstream DNA between -439 and -280 stimulates transcription in an orientation dependent manner. In the TBP promoter region, the upstream DNA between -261 and -207, and the downstream DNA between +15 and +138 are necessary for the stimulation of promoter activity. The upstream DNA (-261 to -207) required for TBP promoter activity contains a 11 bp long palindromic sequence and a DNA replication-related element sequence. Particularly, we could find that the downstream promoter regions of TFIIB and TBP genes contain the conserved nucleotide sequences, suggesting the presence of a common regulatory mechanism for the expression of these two genes.

Animals↗
PubMed1990

Retinoic acid causes a decline in TGF-alpha expression, cloning efficiency, and tumorigenicity in a human embryonal cancer cell line.

The human teratocarcinoma NTERA-2 cl. D1 (NT2/D1) cell is a cloned multipotential embryonal cancer cell line that differentiates into a neuronal phenotype and other cellular lineages with retinoic acid (RA) treatment. Here we report that mRNA for the transforming growth factor-alpha is expressed in these RA-untreated cells and that RA-treatment results in a reduction of mRNA expression within 24 hr of treatment. In total cellular RNA, TGF-alpha mRNA is not detectable by Northern analysis at 6 days when there is increased expression of the human homeotic genes Hu-1 (Hox 2.1) and Hu-2 (Hox 2.2), known markers of RA response in NT2/D1 cells. RA treatment also causes a marked reduction in cloning efficiency and tumorigenicity of these cells. The addition of TGF-alpha or EGF (epidermal growth factor) protein to RA-untreated NT2/D1 cells augments soft agar cloning under limited fetal calf serum conditions. Blocking monoclonal antibodies directed against the EGF receptor (EGFr) can prevent this augmentation. We conclude that TGF-alpha expression inversely correlates with the state of RA-induced differentiation of this human teratocarcinoma cell and that TGF-alpha and EGF proteins are stimulatory growth factors in NT2/D1 cells under these culture conditions.

Cell Differentiation↗
PubMed1992

Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells.

beta-cell type-specific expression of the upstream glucokinase promoter was studied by transfection of fusion genes and analysis of DNA-protein interactions. A construct containing 1,000 bp of 5'-flanking DNA was efficiently expressed in HIT M2.2.2 cells, a beta-cell-derived line that makes both insulin and glucokinase, but not in NIH 3T3 cells, a heterologous cell line. In a series of 5' deletion mutations between bases -1000 and -100 (relative to a base previously designated +1), efficient expression in HIT cells was maintained until -280 bp, after which transcription decreased in a stepwise manner. The sequences between -180 and -1 bp contributing to transcriptional activity in HIT cells were identified by studying 28 block transversion mutants that spanned this region in 10-bp steps. Two mutations reduced transcription 10-fold or more, while six reduced transcription between 3- and 10-fold. Three mutationally sensitive regions of this promoter were found to bind to a factor that was expressed preferentially in pancreatic islet beta cells. The binding sites, designated upstream promoter elements (UPEs), shared a consensus sequence of CAT(T/C)A(C/G). Methylation of adenine and guanine residues within this sequence prevented binding of the beta-cell factor, as did mutations at positions 2, 3, and 5. Analysis of nuclear extracts from different cell lines identified UPE-binding activity in HIT M2.2.2 and beta-TC-3 cells but not in AtT-20, NIH 3T3, or HeLa cells; the possibility of a greatly reduced amount in alpha-TC-6 cells could not be excluded. UV laser cross-linking experiments supported the beta-cell type expression of this factor and showed it to be approximately 50 kDa in size. Gel mobility shift competition experiments showed that this beta-cell factor is the same that binds to similar elements, termed CT boxes, in the insulin promoter. Thus, a role for these elements (UPEs or CT boxes), and the beta-cell factor that binds to them, in determining the expression of genes in the beta cells of pancreatic islets is suggested.

3T3 Cells↗
PubMed1987

Biosynthesis of reovirus-specified polypeptides. Efficiency of expression of cDNAs of the reovirus S1 and S4 genes in transfected animal cells differs at the level of translation.

Full-length cDNAs of the reovirus serotype 1 Lang strain S1 and S4 genes were cloned in Escherichia coli using bacteriophage M13 and expressed in monkey COS cells under the control of the SV40 late promoter using the eukaryotic expression vector pJC119. The s1-encoded sigma 1 and s4-encoded sigma 3 gene products were expressed in transfected COS cells and were indistinguishable from the authentic sigma 1 and sigma 3 polypeptides synthesized in reovirion-infected COS cells. The relative translational efficiencies of the s1 and s4 mRNAs in transfected COS cells were similar to the efficiencies observed in virion-infected cells; the s4 mRNA was translated approximately five times more efficiently than the s1 mRNA. Our results suggest that the differential translation of the reovirus s1 and s4 mRNAs in vivo may be attributed to intrinsic structural properties of the individual mRNAs and is independent of competition with other viral mRNAs.

Animals↗
PubMed1998

[A preliminary study on the in vitro eukaryotic expression of human factor VIII cDNA].

OBJECTIVE: To evaluate the expression efficiency of a cDNA sequence of human clotting factor VIII (4.7 kb, B domain-deleted) in in vitro systems. METHODS: After insertion of the cDNA into several mammalian expression vectors, such as retroviral vector pMSCV, EB virus-based vector pGRE5.2/EBV and eukaryotic expression plasmid pCI, the expression of these constructs were tested in a variety of cells. RESULTS: All the three kinds of constructs-pCI-VIII, pGRE5.2/EBV-VIII and pMSCV-VIII were able to direct FVIII synthesis in NIH3T3, Hela and Bosc23 cells, respectively, while the pMSCV-VIII and pGRE5.2/EBVVIII produced relatively high levels of FVIII activity (up to 0.7 units/ml and 2.0 units/ml from 24 h to 48 h, respectively, after transfection with lipofectamine). The three forms of pMSCV-VIII vector worked in a similar efficacy in Bosc 23 cells, but this function was not detected in NIH3T3, psi-Crip and GP + E86 as well as 32DC13 cells in a transient transfection assay. Moreover, the NIH3T3 and 32DC13 cells infected with culture supernatant from pMSCV-VIII transfected-Bosc23 cells (as packaging cells) unexpectedly did not produce detectable FVIII activity. CONCLUSION: Apart from the design and construction of vectors, target cell selection may play a crucial role in the efficient expression of the FVIII cDNA.

3T3 Cells↗
PubMed1991

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.

Animals↗
PubMed1998

[Human interleukin-13 cDNA cloning and expression].

OBJECTIVE: To construct an efficient expression system for HIL-13 in prokaryotic cells. METHODS: After Amplified by RT-PCR, The cDNA fragment encoding HIL-13 was inserted into pGEX-2T plasmid and expressed under induced condition. RESULTS: HIL-13 cDNA was obtained. Recombinant plasmid pGEX-2T HIL-13 was constructed and sequenced, the inserted fragment of the recombinant plasmid was confirmed to be HIL-13 cDNA. HIL-13 was expressed as a fusion protein with glutathione-s-transferase (GST-HIL-13, MW = 39,000). CONCLUSIONS: (1) HIL-13 cDNA has been obtained from T lymphocytes. (2) The rate of expression of HIL-13 in prokaryotic cells is about 37%.

Amino Acid Sequence↗
PubMed2001

Mechanisms governing expression of the v-FLIP gene of Kaposi's sarcoma-associated herpesvirus.

Open reading frame 71 (ORF 71) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a death effector domain-containing protein that is homologous to cellular FLIPs (FLICE-inhibitory proteins) and is proposed to inhibit Fas-mediated apoptosis. Transcripts bearing ORF 71 (v-FLIP) sequences are present in all latently infected cells. However, mapping studies reveal these to be bi- or tricistronic mRNAs with ORF 71 located 3' to ORFs 72 (v-cyclin) and 73 (latency-associated nuclear antigen), raising the question of how efficient expression of v-FLIP is achieved. We explored this question by examining the expression of model bicistronic (v-cyclin/LUC) transcripts in which a luciferase (LUC) reporter replaced v-FLIP coding sequences. SLK spindle cells transfected with such constructs efficiently expressed luciferase from the 3' position, and this expression was independent of the expression of the 5' v-cyclin gene. Surprisingly, transcript mapping showed that in these cultures, efficient splicing occurred to remove v-cyclin sequences and generate monocistronic LUC transcripts. Similar splicing events produced monocistronic v-FLIP transcripts in KSHV-infected primary effusion lymphoma cells. However, these RNAs were of low abundance and were inducible by treatment with 12-O-tetradecanoylphorbol-13-acetate. Examination of the more abundant bicistronic latent RNAs revealed the presence of an efficient internal ribosome entry site (IRES) overlapping ORF 72 coding sequences. Thus, two potential mechanisms exist for v-FLIP expression, but the evidence suggests that IRES-mediated internal translational initiation on latent polycistronic mRNAs is the principal source of v-FLIP in latency.

Base Sequence↗
PubMed1985

Expression of a mouse U1b gene in mouse L cells.

A 6.9-kb DNA fragment containing two mouse U1b genes was introduced by cotransfection with the Herpes simplex virus (HSV) thymidine kinase (TK) gene into tk- mouse L cells. The parent Ltk- cells produce primarily U1a RNA and only small amounts of U1b RNA. However, after introduction of exogenous U1b genes by cotransfection these cells express large amounts of U1b RNA. Subcloned DNA fragments containing a single U1b gene and varying amounts of DNA flanking the 5' -end of the gene were introduced into Ltk- cells. All of the constructs containing 400 bp or more upstream of the U1b gene were efficiently expressed. By comparison, a subclone containing only 150 bp of DNA flanking the 5' -end of the U1b gene was not efficiently expressed. The U1b transcripts synthesized in transfected cells were identified by hybrid selection and by precipitation of ribonucleoproteins (RNP) containing U1 RNA with anti-RNP antibody.

Animals↗
PubMed2002

Improved mRNA electroporation method for Xenopus neurula embryos.

Electroporation has led to new approaches to the analysis of gene regulation of the chick embryonic system. However, application of this method to Xenopus, another model organism of embryology, has left many difficulties to be overcome. The specially devised electrodes, the examination of luciferase activities expressed, and the direct visualization of green fluorescence protein allow us to optimize the conditions of electroporation for Xenopusembryos. The use of mRNA rather than DNA improved the expression efficiency 120 times more than for the case of plasmid DNA, and the effect emerged more immediately after electroporation. The noncontact electroporation adopted here caused less damage to cells and tissues than with the needle type electrode, making it practical for efficient application to early embryos. Furthermore, the mRNA electroporation technique is applicable for other systems in which the DNA electroporation has not had any significant effect because of its low expression efficiency.

Animals↗
PubMed1990

Two-step transformation of rat 3Y1 cells by the adenovirus E1A and E1B genes.

The transformation of rodent cells by the adenovirus E1A and E1B genes was very efficient when these genes were physically linked. When they were cleaved, the transformation became very inefficient. To clarify this difference, the chimeric E1B genes in which either the adenovirus enhancer or the human beta-actin promoter was linked to the 5' side of the E1B gene were introduced into rat 3Y1 cells. The saturation density of these cell lines (eB or APrB) was similar to that of parental 3Y1 cells. When eB or APrB cell lines were supertransfected with the E1A gene, discrete dense foci were developed after 5-6 weeks, while the supertransfection of 3Y1 derivative cell lines, in which the enhancer-unlinked E1B gene was introduced, did not develop any dense foci. Analysis of the E1A and E1B transcripts in these cell lines indicated that the E1B gene is efficiently expressed in the presence of the E1A gene products if the enhancer is linked to the E1B gene and that an increased level of E1B proteins is required for an efficient expression of the E1A gene. These results indicated that E1A and E1B genes in separate pieces of DNA are capable of cooperatively transforming 3Y1 cells if appropriate cis-acting elements are attached and high-level expressions are achieved.

Actins↗
PubMed1993

Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs.

Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25-50% of embryos and larvae by using the firefly luciferase and the E. coli beta-galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luciferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.

Animals↗
PubMed1998

Pre-mRNA processing enhancer (PPE) element increases the expression of an intronless thymidylate synthase gene but does not affect intron-dependent S phase regulation.

The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human beta-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless beta-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes.

Animals↗