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At least 613 records · Page 34Linked to original sources
PubMed2002

Failed export of the adrenocorticotrophin receptor from the endoplasmic reticulum in non-adrenal cells: evidence in support of a requirement for a specific adrenal accessory factor.

Difficulty in expressing the adrenocorticotrophin (ACTH) receptor (melanocortin 2 receptor; MC2R) after transfection of various MC2R expression vectors has been experienced by many researchers. Reproducible evidence for expression has been obtained only in the Y6/OS3 corticoadrenal cell lines or in cells expressing endogenous melanocortin receptors. In order to determine the cause of this failure of expression we have undertaken the following studies. An MC2R expression plasmid was constructed in which the green fluorescent protein (GFP) coding region had been added to the C-terminus of the mature protein. Transfection of this plasmid into Y6 cells with a cAMP-responsive reporter plasmid demonstrated normal function of this receptor. Imaging of CHO cells expressing MC2R-GFP revealed perinuclear expression, although a cholecystokinin receptor (CCKR)-GFP construct was efficiently expressed at the cell surface. Y6 cells, in contrast, showed cell surface fluorescence after transfection with MC2R-GFP. Several other cell types showed a similar pattern of GFP distribution characteristic of retention in the endoplasmic reticulum. Counterstaining with an anti-KDEL antibody confirmed this location. Co-expression of the MC2R and the CCKR-GFP did not impair CCKR trafficking to the cell surface, implying a receptor-specific impairment to trafficking in the CHO cell which was absent in the Y6 cell.

Adrenocorticotropic Hormone↗
PubMed2001

[Exploration of high expressions of retroviral vectors containing hFIX minigene in murine myoblast cells].

OBJECTIVE: To explore the effect of different vector structure on hF IX protein expression in murine myoblasts in order to construct more efficient expression vector for my oblast-mediated gene therapy for hemophilia B. METHODS: Three types of retroviral vectors pLMe2 IX m(2)SN (forward Me2: Me2 inserted into enhancer region of 3'LTR in forward orientation), pLMe2 IX m(2)SN (reversed Me2) and pL IX m(2)SN which contained intron m(2) and two copies of MCK enhancer (Me2) were transferred into packaging cell line PA317. The hFIX expression level in selected single and mixed clones of stably transfected myoblasts were determined by ELISA and PCR. RESULTS: There was statistically significant difference in hFIX expression levels among the four vectors tested. The expression order was LMe2 IX m(2)SN (forward) > LMe2 IX m(2)SN (reversed) > L IXm(2)SN > L IX SN. The hF IX expression level of forward Me2 vector was higher than that of reversed one, because of one or two copies of reversed Me in the 5'LTR being deleted in the genome of myoblasts, but the orientation of Me or Me2 remained unchanged. CONCLUSIONS: To select a tissue-specific enhancer and optimize FIX minigene construction are effective methods for increasing the expression level. Forward Me2 vector can express hFIX more stably than the reversed one.

Animals↗
PubMed2005

[Preparation of human recombinant kringle 1-5 and its bioactivity].

To investigate antiangiogenesis activity and effects on endothelial cell proliferation of human recombinant K1-5 expressed in E.coli BL21, the cDNA of human K1-5 obtained from a cloning vector pUC57K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E.coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed and purified to a purity of 96% by the nickel affinity chromatography. Refoled K1-5 protein was tested on chicken CAMs, and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, human recombinant K1-5 potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrated that human recombinant K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.

Angiogenesis Inhibitors↗
PubMed1989

Complete structure of the mouse mast cell receptor for IgE (Fc epsilon RI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells.

The high affinity receptor for IgE (Fc epsilon RI) found on mast cells and basophils is a tetrameric complex of a single alpha subunit, a single beta subunit, and two identical gamma subunits. The genes for the three subunits of mouse Fc epsilon RI have now been cloned from the mast cell line, PT18. When compared at the DNA level, the rat and mouse subunits are similarly conserved. However, at the protein level the homology between mouse and rat alpha is surprisingly low (71% identities) especially in the cytoplasmic regions (57% identities) which are of different length (25 and 20 residues, respectively). By contrast the beta and gamma are homogeneously conserved between mouse and rat (83 and 93% identities, respectively). The consensus amino acid sequence of the alpha subunit derived from three species (rat, mouse, and human) shows that the cytoplasmic tail diverges to the same extent as the leader peptide. Conversely, the transmembrane domain of the alpha is highly conserved and contains 10 consecutive residues that are identical. Comparisons between mouse Fc epsilon RI and other mouse proteins reveal regions of high homology between the alpha subunit and Fc gamma RIIa and between the gamma subunit and the zeta chain of the T cell receptor. Cells transfected with the alpha gene express the alpha subunit on their surface very inefficiently. Efficient expression is only achieved after co-transfection of the three rodent genes or of the human alpha gene together with the rodent gamma without apparent need for beta. The subunits are completely interchangeable upon transfection so that various chimeric mouse-rat-human receptors can be expressed.

Amino Acid Sequence↗
PubMed1997

Temperature-induced expression of human-mouse chimeric Fab.

The temperature-induced expression vector pHZ01 with lambda PRPL promoter for the efficient expression of human-mouse chimeric Fab was constructed. Three kinds of chimeric Fab were expressed in E. coli: anti-prostate specific antigen (PSA) chimeric Fab, anti-lysozyme (HEL) chimeric Fab, and anti-tetanus toxoid (TT) chimeric Fab. All the soluble chimeric Fabs expressed showed specific antigen-binding activities.

Animals↗
PubMed2001

Coxsackievirus-adenovirus receptor expression in ovarian cancer cell lines is associated with increased adenovirus transduction efficiency and transgene expression.

The expression of coxsackievirus-adenovirus receptor (CAR) and the integrins alpha(v)beta3 and alpha(v)beta5 was analyzed quantitatively (flow cytometry) and qualitatively (immunocytochemistry) in five human ovarian cancer cell lines (PEO1, PEO4, PEO14, SKOV-3, and OVCAR-3) and three control cell lines (293, HeLa, and CHO-K1). The transduction efficiencies were evaluated by adv/rsv-beta-Gal transduction followed by X-gal staining. The effects of 17beta-estradiol on cell growth, CAR and integrins alpha(v)beta3/5 expression, adenovirus transduction efficiency, and cell-killing efficacy of adv/rsv-tk plus ganciclovir were determined. The levels of CAR, integrin alpha(v)beta3, and integrin alpha(v)beta5 showed great variation between the cell lines. Whereas the expression of CAR appeared to be essential for and positively correlated with adenovirus transduction efficiency, the integrins alpha(v)beta3 and alpha(v)beta5 were not absolutely necessary for adenovirus transduction even though their presence may facilitate transduction. In PEO4 and PEO1 cells, proliferation was stimulated by 17beta-estradiol in a dose-dependent manner. In PEO4 cells, and much less pronounced in PEO1 cells, this was accompanied by an increase in CAR expression. The stimulation of CAR expression was paralleled by an increased transduction efficiency resulting in an increased cell-killing efficacy. Our data suggest that the expression of CAR is one of the most important prerequisites for successful adenovirus-mediated gene therapy of ovarian cancer.

Adenoviridae↗
PubMed1998

An expression system of rat calmodulin using T7 phage promoter in Escherichia coli.

An efficient expression system of rat calmodulin in Escherichia coli is presented. To express rat calmodulin cDNA, we employed a pET expression vector which contains the T7 phage promoter and terminator. After transformation of E. coli BL21(DE3) strain which carries T7 phage RNA polymerase inducible with isopropyl-beta-D-thiogalactopyranoside, induction of the expression, and chromatography of soluble proteins on a phenyl-Sepharose column, about 250 mg of recombinant rat calmodulin was obtained from 1 liter of E. coli culture. The recombinant calmodulin lacked the N-terminal methionine, and posttranslational modifications such as Nalpha-acetylation and methylation. This system facilitates the large amount preparation of calmodulin and the mutant proteins required for the structural analysis by NMR spectrometry and/or X-ray crystallography.

Animals↗
PubMed1998

Innovative and economic potential of mammalian cell culture.

Innovations for economic optimization of manufacturing processes of mammalian cell culture processes address new expression systems, optimized cell culture media and feeding systems, economy of scale, efficient harvest systems for viable cell separation, redesign of downstream processing and reduction of the overall number of quality control assays. A very efficient expression system in Chinese hamster ovary cells is the NEOSPLA expression system yielding 60-100 micrograms monoclonal antibodies per cell and day. Efficient supplements in nutrient feeding are insulin and amino acids which contribute to a high extent to the productivity of the mammalian cell culture process. Large scale manufacturing processes lower cost of goods by reduction of turn around cost for cleaning and steaming in place, media preparation, number of batches for annual campaign, in-process and quality control. In downstream processing the number of process steps and the step yield are responsible for the economics. Process control systems in a computer assisted manufacturing plant increase success rate, reduces man power and minimizes shifts. In the innovative process also alternative technologies such as transgenic animals should be considered to improve the economy of the manufacturing processes.

Animals↗
PubMed1997

In vivo analyses of upstream promoter sequence elements in the 5 S rRNA gene from Saccharomyces cerevisiae.

Upstream promoter elements of the Saccharomyces cerevisiae 5 S rRNA gene have been characterized by genomic DNase I "footprinting" and by in vivo mutational analyses using base substitutions and deletions. A high copy shuttle-vector was used to efficiently express the mutant 5 S rRNA genes in vivo and a structural mutation in the 5 S rRNA, which was previously shown to be functionally neutral but easily detected by gel electrophoresis, allowed for an accurate measure of gene expression. The results provide direct evidence for upstream regulatory elements which confirms a start site element (sse) from -1 to -8 and identifies a new independent upstream promoter element (upe) centered from about -17 to -20. In contrast to previous reports with reconstituted systems, both elements dramatically affect the efficiency of gene expression and suggest that the saturated conditions which are used in reconstituted studies mask sequence dependence; a dependency that could be physiologically significant and play a role in the regulation of 5 S rRNA expression. The footprint analyses support an extended region of protein interaction as recently observed in reconstituted systems but again provide evidence of significant structural rearrangements when the upstream sequence is changed.

Base Sequence↗
PubMed1995

The human beta 2-adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties.

We have developed a rapid and efficient expression system to study the human beta 2 adrenergic receptor (hu beta 2AR) in the fission yeast Schizosaccharomyces pombe. This was achieved by cloning the hu beta 2AR gene, modified by replacement of the 5' untranslated and a small part of the N-terminal coding sequence (first 14 amino acids) with the corresponding region of the yeast Saccharomyces cerevisiae STE2 (alpha-factor receptor) gene. The gene was then placed under the control of a S. pombe constitutive promoter for alcohol dehydrogenase (adh). Hu beta 2AR expression was assessed by immunoblot analysis of the chimeric protein with an anti-STE2 serum raised against a dodecapeptide homologous to the N-terminal amino acids of STE2 and ligand binding was assayed using [125I]cyanopindolol. We demonstrate here that the chimeric receptor expressed in S. pombe exhibits the same characteristic ligand specificity and affinity as that of the authentic hu beta 2AR. This system constitutes a convenient alternative to existing methods for studying seven transmembrane domain receptors due to its simplicity and high reproducibility.

Base Sequence↗
PubMed1997

Heterologous protein production in Acremonium chrysogenum: expression of bacterial cephalosporin C acylase and human thrombomodulin genes.

We have developed an efficient expression system for foreign genes in Acremonium chrysogenum. After inserting the foreign gene between the phosphoglycerate kinase (PGK) promoter and a terminator derived from A. chrysogenum, multiple copies of this expression unit are tandemly ligated into cosmids and the resultant cosmids are introduced into A. chrysogenum. We expressed Pseudomonas cephalosporin C acylase and a human thrombomodulin mutant protein containing the fourth, fifth, and sixth epidermal growth factor (EGF)-like structures (E456). The acylase activity in the transformants obtained using our system was several times higher than that in the transformants without the use of the system. The acylase proteins expressed had enzymatic and immunochemical properties identical to those of authentic acylase. The transformants with the expression plasmid for E456 secreted biologically active E456 protein into the culture medium. The amino terminal sequence of the purified E456 was identical to that of recombinant E456 obtained using mammalian cells.

Acremonium↗
PubMed2003

Expression of transgenes in primary neurons from chick peripheral and central nervous systems by retroviral infection of early embryos.

Many cell biological studies require the expression of transgenes in cells in culture, but it is difficult to obtain uniform, stable, and efficient expression of transgenes in primary neurons. We have approached this problem by adapting from developmental biologists the avian retroviral vector, RCAS. This vector allows the introduction of a transgene by infection early in chick embryonic development. Transgenes that are less than 2.6 kb in size can be cloned through an adapter vector, SLAX 12 NCO, and into the RCAS retroviral vector with relative ease. The vector is then used to produce active virus, and the virus is injected into the neural tube or ventricles of stage 10 embryos. By infecting the neuronal precursor cells while they are still mitotic, the retrovirus and accompanying transgene are introduced into the genome and subsequently spread by replication, shedding of new virus, and infection of other cells. Embryos are incubated from the time of injection until E9-E12 and peripheral and central nervous system neurons are dissected out and grown in culture using standard techniques. In this manner, the majority of the sympathetic and dorsal root ganglion neurons can be induced to express the trangene. A similar result, at lower efficiencies, is obtained for central nervous system neurons.

Animals↗
PubMed2002

Alanine scanning mutagenesis of CCR3 reveals that the three intracellular loops are essential for functional receptor expression.

Intracellular signaling mediated by the eotaxin receptor, CCR3, has been implicated in allergic diseases involving the recruitment and activation of eosinophils. To investigate the structural requirements of the three intracellular loops (ICL) of CCR3, a panel of 15 alanine triplet mutants were generated and their effects on function assessed by assays of cell surface expression and chemotactic responsiveness. While the majority of constructs were efficiently expressed when compared with their wild-type counterpart, their abilities to migrate in response to eotaxin were relatively poor, suggesting that all three intracellular loops of CCR3 are involved to some degree in coupling to G proteins. - Another panel of 7 point mutants were then constructed to examine the DRY motif which resides in ICL2 and is highly conserved throughout the chemokine receptors identified to date. The conservative mutants D130E and R131K were well tolerated and gave chemotactic responses approaching 35 % of wild-type CCR3, but the less conserved substitutions D130A, D130N and R131L were non-functioning. Tyrosine 131 was particularly sensitive to mutation as both Y131F and Y131S mutants were poorly expressed and were chemotactically inactive. Together, this data suggest that the acidic / basic / polar nature of the DRY motif is a prerequisite for CCR3 function.

Amino Acid Motifs↗
PubMed1996

Expression and codon usage optimization of the erythroid-specific transcription factor cGATA-1 in baculoviral and bacterial systems.

Biochemical characterization of cGATA-1, a key transcription factor in the regulation of globin expression in chickens, has been precluded by the unavailability of appreciable amounts of the pure protein. Purification directly from embryonic red blood cells has been limited by the difficulty in obtaining large quantities of the starting material, and previous attempts at bacterial expression have consistently yielded truncated product. To solve these problems, we have taken two approaches to the expression of cGATA-1. First, we were able to produce efficient expression from baculovirus-infected insect cells. Second, by altering the codon usage in cDNA encoding the protein's carboxy-terminal region, we obtained good expression of full-length protein in Escherichia coli. These preparations should prove useful in biochemical and structural studies of the factor. Additionally, we describe a primer extension/PCR-based method which can be used to synthesize extended regions of DNA sequence for gene construction.

Animals↗
PubMed1992

Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans.

The alpha-amylase gene (amy) from Streptomyces griseus IMRU 3570 and the beta-galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P2 or P3. The alpha-amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum.

Amino Acid Sequence↗
PubMed1989

Regulatory studies on the promoter for a gene governing synthesis and assembly of the spore coat in Bacillus subtilis.

gerE is a regulatory gene of Bacillus subtilis that governs the synthesis and assembly of the spore coat and is required for the production of spores that are lysozyme-resistant and germination-proficient. We report the identification of the promoter for gerE and studies on the regulation of its expression. We show that gerE is switched on at the fourth hour of sporulation (stage-V) and that this expression is restricted to the mother-cell chamber of the sporangium. Dependency studies in which the level of gerE expression was measured in 36 different developmental mutants indicate that efficient expression of gerE requires the products of almost all spo0-IV genes tested as well as certain spoV genes. On the basis of its time of induction, compartmentalization of expression and pattern of dependence on other spo genes, gerE is inferred to be regulated co-ordinately with the previously studied spore coat protein gene cotA. gerE and cotA may be members of a developmental regulon of genes whose products are involved in the assembly of the spore coat.

Bacillus subtilis↗
PubMed2003

The role of the MEIS homeobox genes in neuroblastoma.

We recently found amplification of the TALE homeobox gene MEIS1 in the IMR32 neuroblastoma cell line. We now demonstrate high-level expression of the MEIS1 and MEIS2 genes, as well as efficient expression of most other TALE family member genes in a panel of neuroblastoma cell lines. Stable transfection of MEIS1-expressing cell lines with cDNA encoding a naturally occurring dominant-negative splice variant of MEIS1 (MEIS1E) yielded clones with impaired cell proliferation, gain of differentiated phenotype, and increased contact inhibition and cell death. This indicated a relevance of MEIS expression for neuroblastoma cell growth and proliferation. We therefore determined the gene expression profiles of several MEIS1E transfectants using serial analysis of gene expression (SAGE). A large number of genes showed differential expression as a result of MEIS1E expression. These include genes involved in developmental signalling pathways, chromatin binding, cell cycle control, proliferation, and apoptosis. The results presented provide important clues for the oncogenic function of MEIS1 in neuroblastoma.

Chromosomes, Human↗
PubMed2000

A four-nucleotide translation enhancer in the 3'-terminal consensus sequence of the nonpolyadenylated mRNAs of rotavirus.

The 5' cap and poly(A) tail of eukaryotic mRNAs work synergistically to enhance translation through a process that requires interaction of the cap-associated eukaryotic initiation factor, eIF-4G, and the poly(A)-binding protein, PABP. Because the mRNAs of rotavirus, and other members of the Reoviridae, contain caps but lack poly(A) tails, their translation may be enhanced through a unique mechanism. To identify translation-enhancement elements in the viral mRNAs that stimulate translation in vivo, chimeric RNAs were prepared that contained an open reading frame for luciferase and the 5' and 3' untranslated regions (UTRs) of a rotavirus mRNA or of a nonviral mRNA. Transfection of the chimeric RNAs into rotavirus-infected cells showed that the viral 3' UTR contained a translation-enhancement element that promoted gene expression. The element did not enhance gene expression in uninfected cells and did not affect the stability of the RNAs. Mutagenesis showed that the conserved sequence GACC located at the 3' end of rotavirus mRNAs operated as an enhancement element. The 3'-GACC element stimulated protein expression independently of the sequence of the 5' UTR, although efficient expression required the RNA to contain a cap. The results indicate that the expression of viral proteins in rotavirus-infected cells is specifically up-regulated by the activity of a novel 4-nt 3' translation enhancer (TE) common to the 11 nonpolyadenylated mRNAs of the virus. The 4-nt sequence of the rotavirus 3' TE represents by far the shortest of any of the sequence enhancers known to stimulate translation.

3' Untranslated Regions↗