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Results for “expression efficiency”

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At least 469 records · Page 26Linked to original sources
PubMed2003

High-throughput expression, purification, and characterization of recombinant Caenorhabditis elegans proteins.

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.

Animals↗
PubMed2004

Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of alpha-amylase gene from Bacillus amyloliquefaciens.

Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).

Bacillus subtilis↗
PubMed2006

Retroviral vectors encoding a reverse transcription-activated transgene efficiently limit expression of the gene to target cells.

Recombinant retroviral vectors are indispensable tools for the study of gene function and for therapeutic gene transfer owing to their ability to transfer and stably express foreign genes in target cells. A limitation of these vectors, however, is the difficulty in generating stable vector producer cell (VPC) lines when the vectors encode cytotoxic proteins. We developed a series of Moloney murine leukemia virus-based vectors encoding a reverse transcription-activated transgene. These vectors preclude gene expression in the producer cells, yet allow lines for transgene expression in target cells. The vectors were generated by cloning the gene of interest in reverse orientation either just upstream of the viral 3' long terminal repeat (LTR) or in the U3 region of the 3'LTR. An exogenous promoter was inserted, also in reverse orientation, at the R-U5 border of the viral 5'LTR. Upon transduction of target cells, the inserted promoter is copied to the 3'LTR during reverse transcription of the vector genomic RNA, where it then drives transgene expression. We tested this system using a green fluorescent protein (GFP) gene and the SV40 promoter. Reverse transcription-activated retroviral vectors may allow for the generation of stable retroviral VPC lines encoding cytotoxic or inhibitory genes.

Cell Line↗
PubMed2001

Efficient eukaryotic expression system for authentic human sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) is the main carrier for androgens and oestrogens in humans. It mediates the transport of steroid hormones in the circulation and testicular fluid, and regulates their bioavailability to steroid-responsive tissues. In addition, the protein interacts with membrane receptors expressed in target tissues. Binding to the receptors is suspected to facilitate the uptake of steroid hormones and/or elicit cellular signal transduction. The identity of the SHBG receptor has not yet been resolved, in part due to a lack of sufficient quantities of authentic SHBG for receptor purification and molecular characterization. We have successfully addressed this problem by establishing an episomal expression system in human embryonic kidney cells that produces 5 mg of fully active human SHBG per litre. The recombinant protein resembles native SHBG in terms of structure, glycosylation pattern and steroid-binding activity. Moreover, the protein interacts with plasma membranes in steroid target tissues, an activity not observed with SHBG from other recombinant expression systems. Thus our studies have removed an important obstacle to the further elucidation of the role SHBG plays in steroid hormone action.

Cell Line↗
PubMed2006

Efficient constitutive expression of Bacillus subtilis xylanase A in Escherichia coli DH5alpha under the control of the Bacillus BsXA promoter.

Xylanase A (XynA) is a class G/11 xylanase secreted by Bacillus subtilis. XynA was purified to homogeneity from B. subtilis strain 168 culture supernatants by ethanol precipitation and cation-exchange chromatography. The DNA fragment encoding the XynA together with the BsXA promoter region was amplified by PCR from B. subtilis 168 genomic DNA, and cloned into the plasmid pT7T3 to give the plasmid pT7BsXA. After transformation of Escherichia coli DH5alpha with pT7BsXA, a 19-fold increase in the levels of the secreted XynA was detected in the supernatant as compared with the B. subtilis culture. Correct post-translation modification of the recombinant protein was confirmed by N-terminal amino acid sequencing and MS analyses. The pH- and temperature-dependences of the native and recombinant proteins were identical, indicating that the pT7BsXA may be useful for the constitutive expression of heterologous protein in E. coli.

Amino Acid Sequence↗
PubMed1999

A novel system for efficient gene expression and monitoring of bacteria in aquatic environments.

In a previous study, we reported the identification of Escherichia coli genes with increased expression in an aquatic environment. Here, we describe the use of one of these genes, gapC, as an expression system in freshwater habitats. We have identified the transcriptional start site of gapC and analysed the synthesis of the GapC protein during incubation in aquatic medium. The promoter of gapC was used to construct fusions to the reporter genes lacZ and gfp. Analysis of these fusions indicates the potential of gapC as a valuable tool for the detection of E. coli in freshwater habitats, as well as for expressing other genes in aquatic environments.

Base Sequence↗
PubMed2005

Three dileucine-like motifs within the interbox1/2 region of the human oncostatin M receptor prevent efficient surface expression in the absence of an associated Janus kinase.

The oncostatin M receptor (OSMR) is part of receptor complexes for oncostatin M and interleukin-31. Signaling events are triggered by Jaks (Janus kinases) that constitutively bind to membrane-proximal receptor regions. Besides their established role in signaling, Jaks are involved in the regulation of the surface expression of several cytokine receptors. Here, we analyzed the structural requirements within the human OSMR that underlie its limited surface expression in the absence of associated Jaks. We identified three dileucine-like motifs within the Jak-binding region of the OSMR that control receptor surface and overall expression. A receptor mutant in which all three motifs were mutated to alanine displayed markedly increased surface expression. Although the surface half-life of this mutant was increased compared with that of the wild-type receptor, no difference in the internalization rate was detectable, implying that these receptors differ in their post-endocytic fate. The protein stability of the wild-type receptor was markedly lower than that of mutant receptors, but could be strongly increased in the presence of the lysosomal inhibitor chloroquine. Our data are consistent with the dileucine motifs being involved in destabilization of receptors devoid of associated Jaks as part of a quality control ensuring signaling competence of OSMRs.

Amino Acid Motifs↗
PubMed1995

Transduction of genes coding for a histocompatibility (MHC) antigen and for its physiological inducer interferon-gamma in the same cell: efficient MHC expression and inhibition of tumor and metastasis growth.

The mouse mammary carcinoma TS/A, of BALB/c (H-2d) origin, was transfected with the murine interferon-gamma (IFN-gamma) gene (Int. J. Cancer 55: 320, 1993). We used IFN-gamma transfectants as recipients for a second round of transfections with murine allogeneic class I histocompatibility (H-2b) genes that are modulated by IFN. Transfectants with either gene alone, as well as parent TS/A cells (TS/A-pc), were used as controls. Only double transfectants expressed high levels of the allogeneic H-2b genes, while in H-2b single transfectants the expression was very low (but was induced by treatment with exogenous IFN-gamma). The tumorigenic potential of IFN-gamma or H-2b single transfectants was reduced in comparison to TS/A-pc. IFN-gamma+H-2Kb double transfectants were almost nontumorigenic, while IFN-gamma+H-2Db clones gave rise to tumors in about one-half of mice. The experimental metastatic ability of all IFN-gamma+H-2b double transfectants was very low. IFN-gamma single transfectants were known to induce a strong macrophage response in the host. The expression of allogeneic H-2 antigens added a T-lymphocyte-mediated response that accounted for the lower tumorigenicity of double transfectants. These results show that it is possible to steer the immune response evoked by tumor cells for therapeutic purposes. Moreover, the high H-2 expression obtained in IFN-gamma+H-2b double transfectants suggests that single IFN-gamma transfectants are ideal recipients for all IFN-sensitive genes. This approach can be used also for other general-purpose inducers of gene expression.

Animals↗
PubMed1991

Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication.

The product of the rep gene of ColE2 is required for initiation of ColE2 DNA replication. The rep gene was placed under the control of the promoters, PL and PR, and the heat-labile cl857 repressor of bacteriophage lambda. The Rep protein was identified as a 35 Kd protein by the maxicell method in combination with heat-induced expression. The protein was efficiently expressed from these promoters in unirradiated cells and accumulated up to a few per cent of the total cellular proteins. It was partially purified (about 80% pure) and its properties examined. The amino acid sequence of the amino terminal portion of the partially purified protein agreed well with that predicted from the nucleotide sequence of the rep gene. One of the characteristic features of the rep gene is frequent usage of rare codons, especially those for arginine. The protein specifically stimulated replication of ColE2 DNA but not that of ColE3 DNA in crude cell extracts of Escherichia coli. Specific binding of the protein to plasmid DNA containing the origin region of ColE2 was demonstrated by the filter binding method. Neither endonuclease activity nor topoisomerase activity was detected by using ColE2 DNA.

Amino Acid Sequence↗
PubMed1998

Evaluation of hemagglutinin protein-specific immunoglobulin M for diagnosis of measles by an enzyme-linked immunosorbent assay based on recombinant protein produced in a high-efficiency mammalian expression system.

Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.

Adolescent↗
PubMed2001

A simple, rapid, and highly efficient gene expression system for multiheme cytochromes c.

The genes of tetraheme cytochrome c3 and hexadecaheme high-molecular-weight cytochrome c from Desulfovibrio vulgaris could be overexpressed as holoproteins in Shewanella oneidensis TSP-C using pUC-type vectors of E. coli. Surprisingly, S. oneidensis was transformed directly by pUC-type vectors through electroporation. The yields of the recombinant proteins in this expression system were much higher than the previously reported ones.

Cytochrome c Group↗
PubMed2004

A new plasmid-mediated approach to supplement somatotropin production in pigs.

Tremendous progress has been made in the identification of the stimulatory molecules that regulate growth, the mechanisms of action, and the potential application of these molecules for livestock production. A parallel and significant effort is now focused on the discovery and development of economically feasible gene delivery technologies. Plasmid-mediated GHRH gene transfer has emerged as an excellent candidate for agricultural applications to optimize production and animal welfare. We have engineered a GHRH-expressing plasmid that is efficiently expressed in skeletal muscle following intramuscular injection enhanced by electroporation. The GHRH is synthesized in the injected muscle, from which it is secreted to circulate and stimulate normal pituitary GH production and release. Young pigs directly injected with as little as 0.1 mg of a GHRH-expressing plasmid had greater (P < 0.01) weight gain than controls, and a increase (P < 0.05) in fat-free mass. We also have demonstrated that the offspring of gilts injected intramuscularly at d 85 of gestation with a GHRH-expressing plasmid have optimized growth characteristics due to both improved intrauterine weight gain and enhanced maternal lactation performance. Thus, the piglets from treated gilts were larger at birth and weaning compared to controls and reached market weight earlier (P < 0.001). Additionally, pituitaries collected from this group contained an increased number of somatotrophs and lactotrophs (P < 0.001) at birth and at 100 kg. An additional advantage of administering the GHRH plasmid to the gilt compared with the administration of growth-promoting agents to the individual adult animal is a substantial decrease in offspring morbidity and mortality (P < 0.01), which has always represented a major economic loss for the swine industry.

Animals↗
PubMed2004

[Efficient soluble expression, purification and identification of the truncated SAG1 gene of Toxoplasma gondii in Escherichia coli].

OBJECTIVE: To express truncated SAG1 gene in Escherichia coli to obtain purified recombinant SAG1, and identify the immunoreactivity of the product. METHODS: The plasmid pET-30a(+)-trSAG1 was constructed, which was cut by Nco I and HindIII to obtain truncated SAG1 gene and inserted into pET-32a(+) cut by the same two restriction enzymes. After identification by restriction enzymes, the plasmid pET-32a(+)-trSAG1 was transformed into E.coli BL21, and the soluble product induced by 0.1 mmol/L IPTG for 4 hour before purification with Ni-NTA agarose, followed by identification of the purified recombinant protein with SDS-PAGE, Western-blotting and ELISA. RESULTS: Recombinant pET-32a(+)-trSAG1 was successfully constructed and high level of truncated SAG1 expression achieved in E.coli. SDS-PAGE showed that the expressed protein was approximately 40,000 in size and expressed in a soluble form that could be easily purified by Ni-NTA agarose. Western-blotting demonstrated that the purified product could be specifically recognized by sera from rabbits immunized with native antigen of T.gondii, and could also be recognized, as shown by ELISA, by sera from human with T.gondii infection. CONCLUSIONS: The truncated SAG1 gene was successfully expressed in E.coli in a soluble form, and the recombinant protein may be of value in the diagnosis of toxoplasmosis.

Animals↗
PubMed2004

[Expression of human tissue-type plasminogen activator in cow mammary gland].

OBJECTIVE: To construct an expression vector for highly efficient expression of tissue-type plasminogen activator (t-PA) confined in the mammary gland of cow to develop a cow mammary gland bioreactor. METHODS: RT-Touch down-PCR was employed to amplify human tissue-type plasminogen activator (t-PA) cDNA, which was digested with the restriction enzymes and subsequently cloned into the vector pSP72 for constructing specific fusion gene only expressed in the mammary gland. The fusion gene was then transferred into the mouse zygote and the mammary gland tissue of mice and cows. RESULTS: t-PA was detected in the milks of mice and cows after the transgenic manipulation with microinjection and mammary gland injection of the fusion gene. CONCLUSIONS: The vector we constructed can effectively induce t-PA expression in the mammary gland, which is not influenced by different transgenic methods. The expression level of t-PA, however, is significantly higher in the milk of cows than in the milk of mice, suggesting the species-specific difference in milk protein regulating system possibly is due to different factors and regulatory systems.

Animals↗