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At least 415 records · Page 23Linked to original sources
PubMed1998

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

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

3T3 Cells↗
PubMed1991

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

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

Animals↗
PubMed1998

[Human interleukin-13 cDNA cloning and expression].

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

Amino Acid Sequence↗
PubMed2001

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

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

Base Sequence↗
PubMed1985

Expression of a mouse U1b gene in mouse L cells.

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

Animals↗
PubMed2002

Improved mRNA electroporation method for Xenopus neurula embryos.

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

Animals↗
PubMed1990

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

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

Actins↗
PubMed1993

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

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

Animals↗
PubMed1998

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

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

Animals↗
PubMed2004

Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin.

An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.

Animals↗
PubMed2006

High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells.

PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.

Animals↗
PubMed1995

Plasmid insertion vectors that facilitate construction of herpes simplex virus gene delivery vectors.

Plasmid insertion vectors were designed for the expression of foreign genes in recombinant herpes simplex virus (HSV) vectors. One vector, pGal9, was designed for the insertion of foreign genes with their own promoter; a second vector, pGal10, was designed for the insertion of coding sequences downstream from the HSV immediate-early 110K promoter. The 110K promoter directed efficient expression of foreign genes, particularly in replication-incompetent virus recombinants, as shown by the expression of the lacZ and IFN alpha genes. These vectors should be useful for the characterization of various promoters for gene delivery, and for the efficient expression of foreign genes in a variety of cell types.

Animals↗
PubMed1999

[Expression of HBsAg by using various eukaryotic expression vectors].

OBJECTIVE: To establish efficient eukaryotic expression system of HBsAg and its variants. METHODS: A series of expression vectors were constructed by cloning HBV S gene and its mutant genes from recombinant vector pBluescript pBKS(+)-HBs containing S gene and its mutant genes into eukaryotic expression vectors: pMEP4, pCEP4, pLXSN, PXT1, pcDNA3. These recombinant eukaryotic expression vectors were transfected into human hepatocellular carcinoma cells (HepG2). RESULTS: The cell lines expressing recombinant proteins were stably established. CONCLUSION: All expression vectors could express HBsAg and its variants, but their transfecting efficiency, expressing efficiency and stability of expression were different. The establishment of highly efficient expression system in this study paves the way for further study on biologic characters of HBsAg variants, genetic immunization and genetic therapy.

Eukaryotic Cells↗
PubMed2000

Novel trypanosomatid small nucleolar RNAs that guide methylation: their genome organization, expression and potential use to direct specific methylation on target RNA molecules.

Trypanosomatids are the causative agent of several major parasitic diseases including African trypanosomiasis, American trypanosomiasis, and leishmaniasis. These parasites possess unique RNA-processing mechanisms including trans-splicing of pre-mRNA and RNA editing of mitochondrial transcripts. In this study, we identified a trypanosomatid novel group of small nucleolar RNAs that belong to the box C/D snoRNA, which were shown to guide ribose methylation on rRNA. Three snoRNA genes were identified; snoRNA-2 carrying a single snoRNA and g2 and b2 coding for a single or multiple snoRNAs, respectively. Mapping of the methylation sites guided by snoRNA-2 using two different approaches suggest that snoRNA-2 has the potential to guide methylation on both 5.8S and 18S rRNAs. The trypanosomes follow the same guide-methylation rule established for yeast and for mammals. As a first attempt to change the methylation pattern of target RNAs, we generated transgenic parasites carrying the B2 and snoRNA-2, which were engineered to shift the methylation site on rRNA. Despite efficient expression of these tagged snoRNAs, the novel methylation site was not generated. However, efficient expression of tagged snoRNAs in transgenic parasites opens the possibility of engineering novel methylation sites on different target RNAs in vivo.

Animals↗
PubMed2001

Production of recombinant human bile salt-stimulated lipase in Pichia pastoris.

Recombinant human bile salt-stimulated lipase (rhBSSL) was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris. Human BSSL has 16 successively repeated sequences in the carboxy terminal region. The sequence consists of 11 amino acid residues. The coding sequence for the middle 11 of the 16 repeats was removed from hBSSL cDNA to facilitate efficient secretory expression. The clone used for fermentation was a transformant of GS115 (his4) integrated with four copies of the expression cassette containing the modified hBSSL cDNA. Unique fermentation conditions were required for efficient expressions of rhBSSL in the high cell-density fermentation. A sufficient glycerol feed at 30 degrees C and pH 4 under an adequate concentration of dissolved oxygen in the growth phase make the cells active over a long induction period of approximately 15 days. On methanol induction, the concentration of dissolved oxygen should be maintained very low in the presence of sorbitol and skimmed milk at 20 degrees C and pH 5.7. Under these conditions, 0.8-1 g of rhBSSL was secreted in 1 liter of the medium. By immunoelectron microscopy, rhBSSL-tagged gold particles were located in secretion microbodies after the beginning of methanol induction. The secreted rhBSSL was efficiently captured and purified by expanded bed adsorption chromatography.

Cloning, Molecular↗
PubMed1999

TCRzeta is transported to and retained in the Golgi apparatus independently of other TCR chains: implications for TCR assembly.

It is generally assumed that TCR assembly occurs in the endoplasmic reticulum (ER), and ER retention/degradation signals have been identified in several of the TCR chains. These signals are probably responsible for retention of incompletely assembled TCR complexes and free TCR chains in the ER. This study focused on the intracellular localization and transport of partially assembled TCR complexes as determined by confocal microscopy analyses. We found that none of the TCR chains except for TCRzeta were allowed to exit the ER in T cell variants in which the hexameric CD3gammaepsilonTi alphabetaCD3 deltaepsilon complex was not formed. Interestingly, TCRzeta was exported from the ER independently of other TCR chains and was predominantly located in a compartment identified as the Golgi apparatus. Furthermore, in the TCRzeta-negative cell line MA5.8, the hexameric CD3gammaepsilonTi alphabetaCD3 deltaepsilon complex was allowed to exit the ER and was also predominantly located in the Golgi apparatus. However, neither hexameric TCR complexes nor TCRzeta chains were efficiently expressed at the cell surface without the other. The observations that TCRzeta and hexameric TCR complexes are transported from the ER to the Golgi apparatus independently of each other and that these partial TCR complexes are unable to be efficiently expressed at the cell surface suggest that final TCR assembly occurs in the Golgi apparatus.

Biological Transport↗
PubMed1990

Hepatitis B virus (HBV) X gene expression in human cells and anti-HBx antibodies detection in chronic HBV infection.

All mammalian hepatitis B virus genomes contain an open reading frame X (X-ORF) of unknown function which could encode a protein of 17 kDa. Using a plasmid containing the entire X-ORF preceded by the adenovirus type 2 major late promoter and its tripartite leader sequence efficient expression of the HBV X-gene was achieved. The X protein of 17 kDa was characterized by immunoblotting and immunoprecipitated with an antiserum prepared against a X fusion protein produced in E. coli. By cell fractionation and indirect immunofluorescence the X-protein was found at least in part associated with nuclei. Human cell extracts containing the X protein have been used to screen human sera for anti-HBx antibodies. Such antibodies were detected in sera from patients with active chronic hepatitis with ongoing viral replication. The efficient expression of the HBV X protein obtained will facilitate its functional analysis.

Cell Fractionation↗
PubMed2003

[Inhibitive effects of recombinant adenovirus-mediated human endostatin on the growth of human hepatocellular carcinoma xenograft in nude mice].

OBJECTIVE: To investigate the inhibitive effect on the growth of hepatocellular carcinoma (HCC) xenografted in nude mice by adenovirus-mediated human endostatin gene. METHODS: The expression efficiency of endostatin was examined after ECV-304 cells infected with Ad/hEndo by western blot. The hepatoma BEL-7402 cells were injected into Balb/c nude mice to detect the inhibition of Ad/hEndo on the growth of HCC xenografted in nude mice. The expression of endostatin mRNA in tumor tissue was analyzed with RT-PCR, and its distribution in vivo was also analyzed. RESULTS: High level expression of endostatin achieved in infected ECV-304 cells by western blot. Ad/hEndo significantly inhibited the growth of xenografted BEL-7402 tumors (F=4.061, P<0.05). The intratumoral microvessel density (MVD) decreased significantly in the treated mice (6.88+/-1.08 vs 13.60+/-1.71, t=9.216, P<0.01). The expression of endostatin mRNA in tumor tissue was detected by RT-PCR in 3 days after administration intratumorally with Ad/hEndo and almost disappeared in 7 days. Endostatin mRNA was mainly located in tumor tissue with a higher concentration than that in heart, lung, spleen and liver after Ad/hEndo administration. CONCLUSION: Adenovirus-mediated human endostatin gene can be expressed efficiently in vitro and in vivo, and significantly inhibit the growth of BEL-7402 xenografted tumors in nude mice.

Adenoviridae↗