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Results for “expression efficiency”

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At least 811 records · Page 45Linked to original sources
PubMed2006

CBFB-MYH11 hinders early T-cell development and induces massive cell death in the thymus.

Recent studies suggest that the chromosome 16 inversion, associated with acute myeloid leukemia M4Eo, takes place in hematopoietic stem cells. If this is the case, it is of interest to know the effects of the resulting fusion gene, CBFB-MYH11, on other lineages. Here we studied T-cell development in mice expressing Cbfb-MYH11 and compared them with mice compound-heterozygous for a Cbfb null and a hypomorphic GFP knock-in allele (Cbfb(-/GFP)), which had severe Cbfb deficiency. We found a differentiation block at the DN1 stage of thymocyte development in Cbfb-MYH11 knock-in chimeras. In a conditional knock-in model in which Cbfb-MYH11 expression was activated by Lck-Cre, there was a 10-fold reduction in thymocyte numbers in adult thymus, resulting mainly from impaired survival of CD4+CD8+ thymocytes. Although Cbfb-MYH11 derepressed CD4 expression efficiently in reporter assays, such derepression was less pronounced in vivo. On the other hand, CD4 expression was derepressed and thymocyte development was blocked at DN1 and DN2 stages in E17.5 Cbfb(-/GFP) thymus, with a 20-fold reduction of total thymocyte numbers. Our data suggest that Cbfb-MYH11 suppressed Cbfb in several stages of T-cell development and provide a mechanism for CBFB-MYH11 association with myeloid but not lymphoid leukemia.

Animals↗
PubMed2000

Overview of vector design for mammalian gene expression.

The expression of cloned genes in mammalian cells is a basic tool for understanding gene expression, protein structure, and function, and biological regulatory mechanisms. The level of protein expression from heterologous genes introduced into mammalian cells depends upon multiple factors including DNA copy number, efficiency of transportation, mRNA processing, mRNA transport, mRNA stability, and translational efficiency, and protein processing, transport, and stability. Different genes exhibit different rate limiting steps for efficient expression. Multiple strategies are available to obtain high level expression in mammalian cells. This article reviews vector design for expression of foreign genes in mammalian cells.

Animals↗
PubMed1988

Inquiries into the cloning and expression of the tuf gene of Thermus thermophilus HB8.

The tuf gene, which encodes the elongation factor Tu (EF-Tu), of Thermus thermophilus HB8 was cloned and sequenced. The whole G + C content of the tuf gene was 64.9%, and 84.5% of the third base in codon usage was either G or C, in which G was much favorable than C. The tuf gene was expressed in Escherichia coli under the control of the E. coli trp promoter. For the highly efficient expression, the SD sequence of the E. coli trpL was used instead of that of T. thermophilus HB8 and the length between the SD sequence and the initiation codon was controlled to keep 9 nucleotides.

Base Sequence↗
PubMed1998

Enhanced MHC class II-restricted presentation of measles virus (MV) hemagglutinin in transgenic mice expressing human MV receptor CD46.

This study analyzes the role of the measles virus (MV) receptor, i.e. the human CD46 molecule, in the MHC class II-restricted presentation of MV hemagglutinin (H). We generated transgenic mice ubiquitously expressing CD46, with a similar level of transgene expression on the surface of antigen-presenting cells (APC), i.e. B cells, dendritic cells (DC) and macrophages. APC isolated from transgenic mice and nontransgenic controls were tested for their ability to present MV H to H-specific CD4+ I-Ed-restricted T cell hybridomas. All three populations of APC were capable of presenting MV to T cell hybridomas, DC being the most efficient. Expression of CD46 on B lymphocytes increased MHC class II-dependent presentation of MV H up to 100-fold, while CD46-transgenic DC stimulated H-specific T cell hybridomas up to 10-fold better than nontransgenic DC. Interestingly, expression of CD46 did not change the presentation efficiency of transgenic macrophages, indicating that CD46-dependent enhancement of antigen presentation depends on the nature of the APC. Furthermore, a single injection of UV-inactivated MV particles into CD46-transgenic mice, but not nontransgenic controls, induced generation of MV-specific T lymphocytes and production of anti-H antibodies, suggesting a role for CD46 in the efficient capture of MV in vivo. These results show for the first time that one ubiquitously expressed cell surface receptor, like CD46, could function in receptor-mediated antigen presentation both in vitro and in vivo and its performance depends on the type of APC which expresses it.

Animals↗
PubMed1999

Use of carboxypeptidase Y propeptide as a fusion partner for expression of small polypeptides in Escherichia coli.

The carboxypeptidase Y (CPY) propeptide from Saccharomyces cerevisiae was developed as a fusion partner for the efficient expression of small polypeptides in Escherichia coli. Six consecutive histidine residues (6xHis) were fused to the N-terminus of the CPY propeptide for the facilitated purification of fusion proteins using immobilized metal ion affinity chromatography. In addition, a methionine or the pentapeptide (Asp)(4)-Lys linker was inserted at the junction between the CPY propeptide and the target polypeptide to release the target polypeptide by digestion with cyanogen bromide or enterokinase. Therapeutically valuable peptide hormones, such as salmon calcitonin precursor (sCAL-Gly), a fragment of human parathyroid hormone (hPTH(1-34)), and human glucagon were successfully expressed in E. coli as fusion polypeptides with the fusion partner. SDS-PAGE analyses showed that the majority of the expressed fusion sCAL-Gly and fusion hPTH(1-34) were present in the form of inclusion bodies, whereas about 66% of the expressed human glucagon was in a soluble form. Almost complete cleavage of the fusion polypeptides was obtained by digestion with enterokinase. Reverse-phase HPLC analyses showed that the target polypeptides released from the fusion proteins were identical to their native forms.

Calcitonin↗
PubMed2000

GH induced lipolysis stimulation in 3T3-L1 adipocytes stably expressing hGHR: analysis on signaling pathway and activity of 20K hGH.

We have constructed a cell line of 3T3-L1 which can efficiently express human GHR (3T3-L1-hGHR) after differentiation to adipocytes. The expressed hGHR was detected as two bands with approximate molecular sizes of 120K by Western analysis using hGHR specific monoclonal antibody. Maximum lipolytic activity induced by hGH in the 3T3-L1-hGHR was enhanced 10-fold as compared to that in 3T3-L1, suggesting that expressed hGHR is functionally active. Comparative analysis using bGH and hGH revealed that 70% of lipolysis stimulation by 1-10 ng/ml hGH could be attributed to hGHR-mediated response. Analyses on inhibition and phosphorylation of signaling molecules suggested that GH-induced lipolysis stimulation is dependent on gene expression and not mediated through PKA-, PKC-, PLA-, PLC-, nor MAPK-pathway but possibly through JAK-STATs pathway. Duration of STAT5 activation by hGH continued up to 48 h. We also revealed that 22 K hGH isoform, 20K hGH which has been reported as a weaker agonist for GH-induced lipolysis stimulation, possesses equipotent activity and shows stronger action in the presence of hGHBP as compared to 22 K hGH. Taken together we conclude that the hGH-induced lipolysis was not mediated through MAP-, PKA-, PKC-, nor PLA-pathway but might be mediated through STAT pathway and that 20K hGH might show higher lipolytic activity than 22 K hGH in adipose tissue that produces a large amount of GHBP.

3T3 Cells↗
PubMed1994

lacZ expression in germline transgenic zebrafish can be detected in living embryos.

Use of transgenic technology in zebrafish has been limited by the inability to efficiently express transgenes in early embryos of F1 and subsequent generations and to rapidly detect transgenic fish. We generated transgenic fish by injecting fertilized eggs with the Escherichia coli lacZ gene under the control of the Xenopus elongation factor 1 alpha transcriptional regulatory element. Four of five lines of transgenic fish we obtained express the lacZ gene in early embryos. The pattern of expression was distinct for each line, with two lines showing extensive expression beginning at approximately the midblastula transition, one showing patchy expression and one showing expression almost exclusively in motor neurons. Expression patterns were stable through the F2 generation in the three lines studied to date. The availability of these lines facilitated the development of a reliable and rapid method for live-staining lacZ-expressing embryos using the substrate fluorescein-di-beta-D-galactopyranoside (FDG). Positive embryos of the two most highly lacZ-expressing lines could be identified after 2-3 min of staining in FDG and then picked out and raised. These observations should prove useful for a variety of studies in zebrafish.

Animals↗
PubMed2003

Expression and assembly of a fully active antibody in algae.

Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5' and 3' RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

Amino Acid Sequence↗
PubMed1996

Rapid, high-level transient expression of papillomavirus-like particles in insect cells.

Empirical scanning of natural or engineered peptide sequences for functional residues is inherently dependent upon efficient expression of large numbers of individual sequence variants to assay their relative functional potency. The insect baculovirus system has been widely used for expression of viral coat proteins, but it generally requires prior isolation and expansion of a plaque-purified recombinant viral stock to generate useful quantities of self-assembled virus-like particles. In search of a more rapid means of expression of analytical levels of the L1 coat protein of cottontail rabbit and human type 11 papilloma-viruses, we found that even brief transient cotransfection of insect cells with baculovirus plasmid transfer vectors and viral DNA yielded assembled particles that were immunologically indistinguishable from particles obtained with plaque-purified viral stocks. Within six days of plasmid/viral DNA cotransfection of Sf9 cells, at least 1-2 micrograms of assembled L1 particles/100-mm plate could be demonstrated, which proved more than sufficient to assay functionality. Transient cotransfection of insect cells should provide general utility for rapid high-level expression of sets of protein sequence variants, as well as other sequence-scanning applications such as sequence optimization in protein engineering.

Animals↗
PubMed2003

Transgene driving GFP expression from the promoter of the zona pellucida gene zpc is expressed in oocytes and provides an early marker for gonad differentiation in zebrafish.

Although mechanisms of sex differentiation have been studied intensely in mammals, insects, and worms, little is known about this process in lower vertebrates. To establish a marker for female gonad differentiation in zebrafish, we generated a transgenic line in which 412 bp from the promoter and 5' mRNA leader of the female-specific zebrafish zona pellucida gene zpc are fused to the coding region of green fluorescent protein (GFP). The zpc0.5:GFP transgene is expressed exclusively in oocytes, starting from the onset of female-specific differentiation, and closely resembles the expression pattern of the wild-type zpc. Strong GFP expression persists throughout oogenesis and is visible through the body wall of females. We have also characterized a putative upstream factor of zpc, FIGalpha, and show that distribution of FIGalpha RNA is compatible with its postulated role in the regulation of zpc. The zpc0.5:GFP transgenic line described here will be useful for studying oocyte development and the mechanisms that determine sex-specific gene expression in the zebrafish. It is also the first promoter characterized to date to drive stable and efficient expression specifically in the zebrafish female germline.

Amino Acid Sequence↗
PubMed2000

Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage.

We previously cloned a panel of peanut allergens by phage display technology. Examination of the codons used in these sequences indicated that most of the cDNAs contain an excess of the least used codons in Escherichia coli, namely AGG/AGA, that correspond to a minor tRNA, the product of the dnaY gene. To achieve high-level expression of the peanut allergens, the cDNAs were subcloned into an expression vector of the pET series (Novagen) in order to produce (His)(10)-tagged fusion proteins in conventional E. coli BL21(DE3) cells. The peanut allergens Ara h 1, Ara h 2, and Ara h 6 with an AGG/AGA codon content of 8-10% were only marginally expressed, whereas the peanut profilin Ara h 5, with an AGG/AGA codon content of only 0.8%, was efficiently expressed in these cells. Hence, by using modified BL21(DE3) E. coli cells, namely BL21-CodonPlus(DE3)-RIL cells (Stratagene) with extra copies of E. coli argU, ileY, and leuW tRNA genes, it was possible to attain high-level expression of the proteins affected by rare codon usage. IPTG-induced expression of several recombinant peanut allergens, such as Ara h 1, Ara h 2, and Ara h 6, was greatly increased in these special cells compared to the expression yield achieved by conventional E. coli hosts. The purification of the soluble and the insoluble fraction of Ara h 2 was performed by metal-affinity chromatography and yielded a total of about 30 mg (His)(10)-tagged recombinant protein per liter of culture of transformed BL21(DE3)CodonPlus-RIL cells. This is over 100 times more than achieved by production of Ara h 2 in conventional BL21(DE3) cells.

2S Albumins, Plant↗
PubMed2000

Characterization of recombinant mustard trypsin inhibitor 2 (MTI2) expressed in Pichia pastoris.

The mustard trypsin inhibitor MTI2 was expressed as secretory protein in the yeast Pichia pastoris. In order to evaluate the influence of the C-terminal amino acids of the precursor form on the inhibitor activity, the C-terminal precursor and the mature protein were both expressed. A third His-tagged construct was also designed to compare alternative purification procedures. Proteins were efficiently expressed at levels of 40-160 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine beta-trypsin compared to the precursor and His-tagged forms (0.01 nM vs. 0.58 nM and 0.71 nM, respectively). The recombinant proteins were active inhibitors of Spodoptera exigua gut proteases.

Amino Acid Sequence↗
PubMed2002

The circumsporozoite protein of Plasmodium falciparum is expressed and localized to the cell surface in the free-living ciliate Tetrahymena thermophila.

Heterologous expression is an important tool for characterization of protein function, structural studies, and production of antigen. While many different host systems have been utilized for the expression of Plasmodium falciparum proteins, the extreme AT-richness of its genome represents an obstacle to efficient expression. In addition, primary sequence motifs such as glycosyl phosphatidyl-inositol (GPI) cleavage/attachment sites of P. falciparum are not recognized in currently used expression hosts. Recently, DNA-mediated transformation has been used for expression of heterologous genes in the ciliated protozoan Tetrahymena thermophila. We report the stable expression of full-length P. falciparum circumsporozoite (CS) protein in T. thermophila. The expressed gene utilized the native CS protein N-terminal secretory signal sequence and the C-terminal GPI anchoring signal. Immunofluorescence imaging demonstrated that the CS protein was localized to the cell surface of Tetrahymena. Metabolic labeling with tritiated myristate resulted in incorporation of label into the recombinant CS protein, indicating that the protein was bound to the cell surface via a GPI anchor. This is the first report of the recognition of targeting and GPI anchoring signals of the P. falciparum CS protein in a heterologous expression host.

Animals↗
PubMed1999

Detection of placental growth hormone variant and chorionic somatomammotropin-L RNA expression in normal and diabetic pregnancy by reverse transcriptase-polymerase chain reaction.

Diabetes is a common complication encountered during pregnancy. Earlier studies indicated that diabetic placentas bear morphological alterations consistent with modified placental differentiation, including alterations in the villous cellular content, structure, and total surface. Limited data associating the diabetic status with the expression of terminal placental differentiation markers are available. The human growth hormone/chorionic somatomammotropin (hGH/CS) family consists of five genes, one of which (GH-N) is expressed efficiently in pituitary while the other four (CS-A, B, L, and hGH-V) are expressed in placenta and represent ultimate placental differentiation markers. We developed and applied a sensitive RT-PCR method coupled with diagnostic restriction digestion to determine the relative levels of the hGH/CS family in normal pregnancies and examine whether their mRNA expression pattern is altered in pregnancies complicated by diabetes. We show that relative hCS-L content changes during placental development. Specifically, normal term placentas express higher relative levels of hCS-L, lower relative hGH-V levels and a 70-fold lower hGH-V/CS-L mRNA ratio compared to early placentas. Also, many term placentas from diabetic pregnancies express lower relative levels of hCS-L mRNA and a much higher hGH-V/CS-L mRNA ratio compared to normal term placenta, resembling more an early placenta pattern of expression. Thus, our study suggests that the expression of terminal placental differentiation markers, such as the hGH/CS genes, is altered in term placentas from these diabetics reflecting either impaired placental differentiation or post-differentiation impairment of normal placental function.

Diabetes, Gestational↗
PubMed2003

A vector with the downstream box of the initiation codon can highly enhance protein expression in Escherichia coli.

An expression vector, pET-DB, with a perfectly matching downstream box of the initiation codon has been constructed on the basis of the pET system. Any gene of interest can then be inserted into the vector. Four genes were used to test the expression efficiency of the vector. The results show that the vector pET-DB can further increase protein expression level at least up to 35-70% as compared with the initial T7 expression system, indicating that the downstream box can enhance protein expression in Escherichia coli.

Adenylate Kinase↗
PubMed1998

The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells.

Under physiological salt concentration, plasmid DNA complexed with transferrin-conjugated or unmodified polyethylenimine (PEI, 800 kDa) forms huge (up to > 1000 nm) aggregates, unless the individual components are mixed at a highly positive nitrogen/phosphate (N/P) charge ratio. At low ionic strengths, however, small particles with an average size of 40 nm are formed over a broad range of N/P ratios. Interestingly, in transfection experiments these small particles result in a 10-fold (B16F10 cells) to more than 100-fold (Neuro2A cells, K562 cells) reduced luciferase gene expression efficiency in comparison to the large complexes formed in physiological salt solutions. Limited transport of the small particles to the cell surfaces is one possible reason for this effect. Application of the small particles in more concentrated form and over extended periods of time improves transfection activity. Reduced intracellular release may be another explanation for the decreased transfection efficiency; incubation with chloroquine or incorporation of the endosomolytic peptide INF5 into the small complexes enhances gene expression approximately 10-fold. Analysis of gene expression at the cellular level using a green fluorescence protein reporter gene and flow cytometry revealed that the differences in overall gene expression largely result from different intensities per expressing cell, while the difference in the percentage of expressing cells is less substantial.

Animals↗
PubMed2001

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the E3B region.

Successful therapies for cancer need to deal with the complexity associated with the human tumor. Studies of tumor and viral biology have progressed to a point where replicating viruses are now being engineered as potential treatments for human cancers. The complex nature of human cancers dictates that successful treatments will require combination therapies. To this end, we have focused on developing the gene delivery capacity of the replicating adenovirus, using the non-essential E3 region transcription unit as a target site for therapeutic transgene insertions. Utilizing the endogenous expression machinery of the E3 region (promoter, splicing, polyA) we show that a therapeutic transgene, TNF, is efficiently expressed from the E3B region and with exclusive late gene expression kinetics. Potential clinical applications are discussed.

Adenovirus E3 Proteins↗
PubMed2000

Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus: implications for the safety of lentiviral vectors.

The safety of lentiviral vectors for clinical applications is still a major concern. The gag-pol expression plasmids and the lentiviral vectors used in previous studies contain homologous regions, which constitute a risk for recombination events. Synthetic gag-pol genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were therefore constructed, in which the codon usage was optimized for expression in human cells without altering the amino acid sequences. The synthetic gag-pol genes allowed efficient expression of these genes in the absence of Rev and the 5' untranslated leader region. Both the HIV-1 and the SIV synthetic gag-pol expression plasmids could mediate transduction of an SIV vector into nondividing human cells with titers of about 10(6) transducing units/ml. Similar titers were obtained with a four-plasmid vector-packaging system based on HIV-1. Using a biological assay, homologous recombination events between the synthetic gag-pol expression plasmids and an SIV vector were undetectable and in comparison with a previously used gag-pol expression plasmid at least approximately 100-fold less frequent. By eliminating regions of homology and sequences involved in packaging, synthetic gag-pol genes should improve the safety profile of lentiviral vectors.

5' Untranslated Regions↗