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Results for “expression efficiency”

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At least 325 records · Page 18Linked to original sources
PubMed1984

Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins.

Construction of a family of bacterial expression vectors, pEX1-3, is described. These vectors are derived from a cro-lacZ gene fusion plasmid which expresses large quantities of fusion protein under the control of the PR promoter of bacteriophage lambda. A polylinker has been engineered into the 3' end of the lacZ gene in all three translational reading frames, and stop signals for transcription and translation inserted, so that any open reading frame DNA may be expressed as a hybrid beta-galactosidase protein. cDNA fragments cloned in these vectors can be detected with an efficiency of greater than 1 in 3, thus enabling the detection of rare cDNA molecules. In addition, the low solubility of hybrid proteins leads to a rapid isolation procedure allowing antibodies of pre-determined specificity to be made against expressed regions of cloned DNA. We describe the cloning of albumin and complement C9 genes from a human cDNA library using polyclonal and monoclonal antibodies.

Bacteriophage lambda↗
PubMed1983

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.

Animals↗
PubMed2002

A proinsulin gene splice variant with increased translation efficiency is expressed in human pancreatic islets.

As glucose-induced insulin expression is mainly regulated at the translational level, and such regulation often involves the 5'-untranslated region (5'UTR), we examined the human proinsulin gene 5'UTR. RT-PCR and sequencing demonstrated that a proinsulin splice variant (SPV) generated from a cryptic 5'-splice site and retaining the first 26 bp of intron 1 was present in human pancreatic islets from normal donors. The expression of this SPV was metabolically regulated, as shown by quantitative real-time RT-PCR, revealing a more than 10-fold increase in the SPV in isolated human islets incubated at 16.7 mM compared with 1.67 mM glucose. In vitro wheat-germ translation and in vivom transfection studies demonstrated that the altered 5'UTR of the SPV increased translation. The SPV yielded 4-fold more in vitro translated preproinsulin protein than the native proinsulin mRNA, and the SPV 5'UTR inserted upstream from a luciferase reporter gene resulted in a more than 6-fold higher luciferase activity, suggesting enhanced translation in vivo. Retention of the 26 bp changed the proposed secondary RNA structure of the SPV, which may facilitate ribosomal binding and explain the increase in translation efficiency. These results suggest a novel mechanism by which metabolic changes can modulate the expression of 5'UTR SPVs and thereby regulate translation efficiency.

5' Untranslated Regions↗
PubMed1988

Cloning of a LEU gene and an ARS site of Rhodotorula glutinis.

Genomic library of DNA from an extracellular protease-producing yeast strain, Rhodotorula glutinis K-24, was constructed, using Escherichia coli plasmid vector PBR322. A LEU gene from R. glutinis was cloned, and found to complement leu- mutations in E. coli and Saccharomyces cerevisiae. In E. coli, the LEU gene in the cloned yeast DNA fragment was efficiently expressed when inserted into the vector in one orientation. In S. cerevisiae, the R. glutinis LEU gene was efficiently expressed when inserted into a shuttle vector YEP13 in both orientations, suggesting that the isolated R. glutinis DNA fragment contains a promotor sequence of R. glutinis in front of the LEU gene. In addition, our data suggests that the cloned LEU fragment also contains an ARS (autonomously replication sequence) site of R. glutinis that could function in S. cerevisiae.

Blotting, Southern↗
PubMed2002

Cytotoxic immune response blunts long-term transgene expression after efficient retroviral-mediated hepatic gene transfer in rat.

Vectors derived from oncoretroviruses can transduce a small proportion of hepatocytes when injected in the regenerating liver. Transgene expression may be sustained for months without immune response. In striking contrast, we observed a rapid extinction when the intravenous injection of a high input of nuclear beta-galactosidase (beta-gal) expression vector, one day after partial hepatectomy, led to a significant proportion of transduced cells in the liver. Extinction was associated with liver inflammation on tissue sections and appearance of antibodies against the transgene product, while vector genomes became undetectable in liver tissue by PCR. These observations suggested the elimination of transduced cells by an immune response. Transgenic rats tolerant for cytoplasmic beta-gal, or normal rats depleted in CD8 T lymphocytes, steadily expressed the beta-gal vector. In the spleen of normal rats, we detected cytotoxic cells directed against cells expressing beta-gal after the injection of the beta-gal vector. In jaundiced Gunn rats deficient in bilirubin glucuronosyl transferase (BGT1) and treated with a human BGT1 cDNA expression vector, we observed the same kinetics of extinction as well as the appearance of anti-BGT1 antibodies. This study demonstrates that retrovirus-mediated gene transfer may induce cytotoxic T lymphocytes specifically directed against transgene-expressing cells.

Animals↗
PubMed1997

Expression and efficient export of enzymatically active Mycobacterium tuberculosis glutamine synthetase in Mycobacterium smegmatis and evidence that the information for export is contained within the protein.

We have investigated the expression and extracellular release of active, recombinant Mycobacterium tuberculosis glutamine synthetase (EC 6.3.1.2), an enzyme that is a potentially important determinant of M. tuberculosis infection and whose extracellular release is correlated with pathogenicity. The M. tuberculosis glutamine synthetase gene encodes a polypeptide of 478 amino acids; 12 such subunits comprise the active enzyme. Northern blot, nuclease S1, and primer extension analyses revealed glutamine synthetase specific transcripts of approximately 1,550 and 1,650 nucleotides produced under low and high nitrogen conditions, respectively. Expression of recombinant M. tuberculosis glutamine synthetase in Escherichia coli YMC21E, a glutamine synthetase deletion mutant, led to transcomplementation of the mutant but not to release of active enzyme. Expression in Mycobacterium smegmatis 1-2c, from the gene's own promoter, resulted in the release of >95% of all recombinant enzyme. No hybrid molecules containing M. tuberculosis and M. smegmatis glutamine synthetase subunits were detected. Native and recombinant exported and intracellular glutamine synthetase molecules were indistinguishable from one another by mass, N-terminal amino acid sequence, antibody reactivity, and enzymatic activity. Since M. tuberculosis glutamine synthetase is similar to other, strictly intracellular, bacterial glutamine synthetases and the DNA sequence upstream of the structural gene does not encode a leader peptide, the information to target the protein for export must be contained in its amino acid sequence and/or conformation.

Amino Acid Sequence↗
PubMed1995

Analysis of the role of 5' and 3' flanking sequence elements upon in vivo expression of the plant tRNATrp genes.

We have isolated the majority (seven) of the tRNA(Trp) genes of Arabidopsis and have studied the 5' and 3' flanking sequence requirements for their efficient expression in vivo by using an assay requiring translational suppression of the luciferase reporter gene. The expressed tRNA(Trp) genes contain no highly conserved 5' flanking sequences; however, these sequences are distinctly AT rich, contain several possible TATA elements, and are bound in vitro by recombinant plant TATA binding protein. Replacement of the natural 5' flanking sequences with three different sequences lacking TATA elements reduced expression in vivo up to 10-fold; the same effect was observed when the TATA elements of the natural 5' sequences were inactivated by point mutations. Introduction of a single TATA element from the adenovirus major late promoter into an artificial 5' flanking region of the tRNA(Trp) gene enhanced expression in vivo when the TATA element was placed at position -32 relative to the first nucleotide of the mature tRNA sequence, but not when it was placed at position -24. Primer extension analyses of in vitro transcripts revealed that the position of the TATA element helps dictate the start site of transcription. Efficient expression of the tRNA genes in vivo also required 3' flanking sequences capable of terminating transcription.

Arabidopsis↗
PubMed1991

Regulated expression allows high level production and secretion of HIV-1 gp120 envelope glycoprotein in Drosophila Schneider cells.

We have established a stable, continuous culture Drosophila Schneider 2 cell line that efficiently expresses a secreted, truncated form of the HIV envelope gp120 protein in a regulated manner. The Drosophila produced recombinant gp120 protein is highly glycosylated, is recognized by gp120-specific monoclonal antibodies, binds to the CD4 receptor and has the ability to inhibit syncytia formation between uninfected CD4+ cells and HIV infected cells. We conclude that this recombinant Drosophila envelope protein is an appropriate mimic of the authentic viral envelope protein. Thus, the Drosophila cell provides a continuous, stable culture system for the efficient expression of secreted forms of complex surface glycoproteins in quantities sufficient for detailed analyses.

Amino Acid Sequence↗
PubMed2001

Efficient gene expression in skin wound sites following local plasmid injection.

Transfection of the skin by local gene delivery, as well as widespread transfection of systemic tissues following intravenous injection of cationic liposome/DNA complexes have been reported. Here, we show that surgically wounded mouse skin can be transfected either by local injection of DNA alone or by intravenous injection of optimized cationic liposome/DNA complexes; however, direct cutaneous injection produces much higher levels of gene expression in the skin, which is targeted to dermal and subdermal layers. High levels of chloramphenicol acetyltransferase activity were present from 3 h to 2 wk following direct injection of a gene expression plasmid into wounded skin and were maintained at detectable levels up to 8 wk after injection. Expression of transferred chloramphenicol acetyltransferase as well as beta-GAL genes was localized to fibroblasts, macrophages, and adipocytes as determined by histochemistry and immunohistochemistry. Further- more, local injection of a human granulocyte- colony-stimulating factor gene expression plasmid produced high levels of the biologically relevant human granulocyte-colony-stimulating factor protein in wounded mouse skin. This efficient and simple method of site-specific gene transfer into wounds may lead to the development of cutaneous gene therapy directed against disorders of abnormal cutaneous wound healing.

Animals↗
PubMed1998

Structure and promoter analysis of Math3 gene, a mouse homolog of Drosophila proneural gene atonal. Neural-specific expression by dual promoter elements.

ath3, a vertebrate basic helix-loop-helix gene homologous to Drosophila proneural gene atonal, can directly convert non-neural cells into neurons with the anterior features. In the mouse, ath3 expression initially occurs widely in the developing nervous system and then gradually becomes restricted to the neural retina. Here, we characterized the genomic organization and promoter activity of mouse ath3 (Math3). Math3 gene consists of two exons separated by an 8-kilobase intron, and the whole protein-coding region is located in the second exon. Transcription starts at two sites, which are 75 nucleotides apart from each other, and there is no typical TATA box in the upstream region of either start site. Transient transfection analysis showed that the 5'-region of Math3 can direct efficient expression in neuroblastoma cells but not in glioma or fibroblast cells. Deletion studies revealed that the proximal 193-base pair region, which contains the downstream transcription initiation site but not the upstream site, is essential for the Math3 promoter activity and can direct efficient expression in neuroblastoma cells. In contrast, retrovirus-mediated promoter analysis demonstrated that a region further upstream is additionally necessary for retinal expression. These results indicate that Math3 promoter contains two essential regulatory regions, the proximal 193-base pair region, which confers efficient neural-specific expression, and a region further upstream, required for retinal expression.

Animals↗
PubMed1992

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.

Animals↗
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↗