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PubMed2005

[High-level expression of the potential vaccine antigen TSO18 of Taenia solium in Pichia pastoris].

TSO18 gene was subcloned into the Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-TSO18 was transformed into P. pastoris GS115 by electroporation so that the plasmid will be integrated with chromosome of P. pastoris. The P. pastoris strains containing multi-copy recombinant were screened by G418 and induced by methanol. The expression product was analyzed by SDS-PAGE, Western blot, deglycosylation, and purified by Sephadex column, and was used to immunize mice. The results indicated that the target protein was efficiently expressed in P. pastoris, and glycosylated moderately, and had immunological activity. In a 5 liter fermentor, the expression level of the target protein was up to 2.54 mg/mL. These results will benefit for the development of genetically engineering vaccine.

Animals↗
PubMed1991

Post-transcriptional regulation of the human liver/bone/kidney alkaline phosphatase gene.

Osteoblasts express high levels of liver/bone/kidney alkaline phosphatase (LBK AP), an enzyme critical for bone formation. Other tissues and cell types generally express much lower levels of LBK AP and correspondingly lower levels of mRNA. In light of our early observations that the human LBK AP promoter is expressed equally when transfected into a variety of different cells, we have carried out a detailed study of LBK AP gene expression in Saos-2 cells which are osteoblast-derived and express high levels of LBK AP mRNA, and in HepG2 hepatoblastoma cells which express LBK AP mRNA at levels which are approximately 1000-fold lower. Our results indicate that both of these cells utilize the same promoter sequences to initiate transcription of their LBK AP genes at roughly the same rates. Moreover, the stability of cytoplasmic LBK AP mRNA is equal in both cell types. The lack of any apparent buildup of unspliced precursor mRNA in the nucleus of HepG2 cells leads us to the conclusion that splicing (and nuclear export) is equivalent. It is therefore likely that differential expression is controlled at a very early step post-transcription, possibly by sequences that destabilize the nascent RNA in HepG2 cells. We reason that these destabilizing sequences are located in the gene's introns because a transfected LBK AP minigene, comprised of the full length cDNA and flanking sequences, is expressed efficiently in both cell types.

Alkaline Phosphatase↗
PubMed1993

Promoter activity of human renin 5'-flanking DNA sequences is activated by the pituitary-specific transcription factor Pit-1.

Although the renal juxtaglomerular cell is the source of circulating renin, the renin gene is also expressed at a number of extrarenal sites including lactotrope cells of human and ovine pituitaries. In the present study, we demonstrate that GC cells, a pituitary lactotrope precursor cell line, efficiently express transiently transfected hybrid genes containing human renin 5'-flanking DNA sequences -148/+11. Gel mobility shift competition analyses show that a highly conserved sequence in human and rodent renin 5'-flanking DNAs (human coordinates: -80/-58) binds a nuclear factor from GC cells, most likely the pituitary-specific factor Pit-1. Deletional and mutational analyses demonstrate that this site is a major determinant of renin promoter activity in GC cells. Transfection of a Pit-1 expression construct into HeLa cells, where activity of the human renin promoter is low, stimulates expression of cotransfected renin-luciferase constructs. Moreover, activation of the human renin promoter by co-expression of Pit-1 is dependent on an intact Pit-1 site. Taken together, these data strongly suggest that Pit-1 activates pituitary renin gene expression. This finding raises the possibility that member(s) of the POU family of transcription factors, of which Pit-1 is an archetypal member, may direct renin expression in other tissues, including the kidney.

Animals↗
PubMed1997

Ambisense gene expression from recombinant rabies virus: random packaging of positive- and negative-strand ribonucleoprotein complexes into rabies virions.

We have recovered from cDNA a rabies virus (RV) containing identical, transcriptionally active promoters at its genome (negative-strand) and antigenome RNA and directing efficient expression of a chloramphenicol acetyltransferase (CAT) reporter gene from the antigenome. Transcription of the antigenome CAT gene was terminated by a modified RV gene junction able to mediate transcription stop and polyadenylation but not reinitiation of downstream transcripts. While in standard RV-infected cells genome and antigenome RNAs were present in a 49:1 ratio, the ambisense virus directed synthesis of equal amounts of genome and antigenome RNA (1:1). Total replicative synthesis was reduced by a factor of less than 2, revealing an unexpectedly high level of replication activity of the transcriptionally active promoter in the absence of the parental antigenome promoter. Successful packaging of ambisense ribonucleoprotein complexes (RNPs) into virions demonstrated that the parental 5' end of the RV genome RNA does not contain putative signals required for incorporation into virions. As determined both for standard RV and ambisense RV, virus particles contained genome and antigenome RNPs in the same ratios as those present in infected cells (49:1 and 1:1, respectively), indicating indiscriminate incorporation of RNPs independent of signals in the RNA. Ambisense expression of multiple foreign genes from RV vectors may circumvent problems with transcriptional attenuation of rhabdovirus housekeeping genes.

Animals↗
PubMed2007

Oct-4, Rex-1, and Gata-4 expression in human MSC increase the differentiation efficiency but not hTERT expression.

Micro-environment seems to exert an important influence on human mesenchymal stem cell (MSC) differentiation and proliferative capacity in bone marrow as well as in culture ex vivo. Oct-4, Rex-1, and TERT genes are well-known for the maintenance of pluripotentiality differentiation and the proliferative capacity of embryonic stem cells. Some previous data report expression of these embryonic factors in selected clones from bone marrow adult stem cells. Our goal was to study expression of Oct-4, Rex-1, and TERT in primary cultured human MSC according to the serum concentration. In addition, we have studied the expression of Gata-4 since this factor plays a key role in organogenesis. We hypothesized that low serum concentration with appropriate growth factors may induce an undifferentiated status with a re-expression of embryonic factors and extend differentiation capacity. Thus, using a defined culture medium, we report on the increased expression of Oct-4, Rex-1, and Gata-4 in human MSC. We have correlated this expression to an increase in differentiation efficiency towards osteogenic and adipogenic phenotypes. Our data suggest that the culture medium used permits the emergence of adult stem cells with a high differentiation capacity and expression of embryonic factors. These cells may have important implications for cell therapy.

Adipocytes↗
PubMed1983

Gene expression: chemical synthesis and molecular cloning of a bacteriophage T5 (T5P25) early promoter.

A sixty base pair DNA duplex containing the nucleotide sequence of the bacteriophage T5 early (T5P25) promoter has been constructed using a combination of chemical synthesis and enzymatic methods. Subsequent to cloning into pBR322, the promoter has been demonstrated to be biologically active being capable of directing the efficient expression of genes under its control. This serves as a prototype for an approach to the study of the in vivo structure-function relationships and efficiency of promoters.

Base Composition↗
PubMed2001

Plants expressing tomato golden mosaic virus AL2 or beet curly top virus L2 transgenes show enhanced susceptibility to infection by DNA and RNA viruses.

The AL2 gene of the geminivirus tomato golden mosaic virus (TGMV) encodes a transcriptional activator protein (TrAP) that is required for efficient expression of the viral coat protein (CP) and BR1 gene promoters. In contrast, L2, the positional homolog of AL2 in the related beet curly top virus (BCTV), is not required for CP expression, raising questions about the functional relationship between the AL2 and L2 gene products. In this study, transgenic Nicotiana benthamiana and N. tabacum var. Samsun plants expressing a truncated AL2 gene (AL2(1-100), lacking the activation domain) or full-length L2 were prepared. These transgenic plants showed a novel enhanced susceptibility (ES) phenotype following inoculation with TGMV, BCTV, or tobacco mosaic virus (TMV), an unrelated RNA virus. ES is characterized by a reduction in the mean latent period (from 1 to 9 days) and by a decrease in the inoculum concentration required to infect transgenic plants (ID50 reduced 6- to 60-fold). However, ES does not result in an enhancement of disease symptoms, and viral nucleic acids do not accumulate to substantially greater levels in infected transgenic plants. That both viral transgenes condition ES suggests that their products share the ability to suppress a host stress or defense response that acts against DNA and RNA viruses. The data further indicate that the transcriptional activation activity of AL2 protein is not required for suppression. The nature of the response targeted by the AL2 and L2 gene products is discussed.

Geminiviridae↗
PubMed2002

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

Corynebacterium↗
PubMed2000

Bacterial expression and purification of the Arabidopsis NADPH-cytochrome P450 reductase ATR2.

An N-terminally modified form of the Arabidopsis NADPH-cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2',5'-ADP-agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 micromol min(-1) mg(-1) and the K(m) for cytochrome c reduction was 15 +/- 2 microM. The purified NADPH-cytochrome P450 reductase was able to support function of CYP79B2.

Amino Acid Sequence↗
PubMed1999

Expression and secretion of a biologically active mouse sonic hedgehog protein by the methylotrophic yeast Pichia pastoris.

We have successfully secreted the amino-terminal functional domain of mouse sonic hedgehog protein (SHH) into culture fluid using a yeast Pichia pastoris expression system. A cDNA fragment encoding the amino-terminal domain of mouse SHH was inserted downstream of the Saccharomyces cerevisiae alpha-mating factor secretion signal. The DNA fragment was introduced into the host genome by the spheroplast transformation method. Transformants were selected based on their resistance to G418: His+ transformants which showed resistance to over 8 mg G418/ml were selected and analyzed for determination of the plasmid copy number. One His+ clone which has eight copies of the expression cassette per genome was cultured in minimal medium deficient for histidine, and further cultured in buffered medium supplemented with methanol which activates the AOX1 promoter. SDS-PAGE analysis indicated efficient expression and secretion of mouse SHH into culture fluid. The yield of secreted SHH was estimated to be 50 micrograms/ml. Purified protein was assayed for biological activity and found to activate the transcription of the Patched genes (Ptc-1 and Ptc-2) encoding receptors for SHH.

Animals↗
PubMed1994

Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli.

The cDNA encoding mouse phosphatidylinositol transfer protein (PI-TP) was isolated by means of reverse transcriptase polymerase chain reaction. The nucleotide sequence of this cDNA has a high similarity (98%) with that of rat PI-TP; the predicted amino acid sequence is 99.6% identical to that of rat PI-TP. The cDNA encoding mouse PI-TP was cloned into the expression vector pET3d and the Escherichia coli strain BL21(DE3) was transformed with the resulting plasmid. After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside, PI-TP was efficiently expressed in the E. coli strain. It was estimated that 5% of the total soluble cell protein consisted of PI-TP. The recombinant mouse PI-TP was purified from the bacterial lysate in four steps: ammonium sulphate precipitation, anion-exchange chromatography, heparin-Sepharose affinity and gel filtration chromatography. Fractionation on the heparin-Sepharose affinity column yielded two forms: PI-TP Hepa1 and Hepa2. These two proteins have the same molecular mass of 35 kDa, both contain a phosphatidylglycerol molecule and both are recognized by anti-PI-TP antibody. Both recombinant proteins have an isoelectric point of 5.4 as compared to 5.5 for bovine brain PI-TP. Sequence analysis of the first 25 N-terminal amino acid residues showed that both forms are identical, except that PI-TP Hepa1 contains the initiator methionine which is lacking from PI-TP Hepa2. The two PI-TP forms have similar phospholipid-binding and transfer activity, comparable to that of bovine brain PI-TP. Both forms and bovine brain PI-TP are phosphorylated equally well in a Ca2+/phospholipid-dependent way by protein kinase C.

Amino Acid Sequence↗
PubMed1976

Clinical testing of the radiosensitiser Ro-07-0582. II. Radiosensitisation of normal and hypoxic skin.

The effectiveness of the radiosensitiser Ro-07-0582 has been tested in man by observing the skin reaction after irradiation using a radio-strontium source with and without administration of the drug. The skin reaction technique has been extended so that skin was irradiated when at low oxygen tension (hypoxic) as well as when fully oxygenated (oxic). Limbs have been made hypoxic by occlusion of the circulation using a sphygmomanometer cuff. To this has been added the prior use of an Esmarch's bandage to reduce the volume of blood in the skin and the enclosing of the limb in a bag of nitrogen. The Oxygen Enhancement Ratios obtained without the sensitiser in nine studies using this system ranged from 1-64 to 2-46. Using Ro-07-0582 skin reactions after oxic exposures were not enhanced but those after hypoxic exposures were markedly increased. The Relative Sensitising Efficiency expresses, as a percentage, the restoration of the sensitivity of cells protected by hypoxia. In six cases where Ro-07-0582 was given in doses ranging from 81 to 165 mg/kg, efficiencies ranged from 27 to 71%. These results can be compared with three studies using metronidazole where efficiencies ranged from 11 to 15%. Using comparable dosage Ro-07-0582 seems approximately three times more effective than metronidazole. In this first study in man, Ro-07-0582 appears to be a highly effective radiosensitiser of hypoxic cells.

Constriction↗
PubMed2005

Rapid method for the construction of Salmonella enterica Serovar Typhimurium vaccine carrier strains.

Salmonella enterica serovar Typhimurium is a versatile organism for the generation of live recombinant vaccines for mucosal immunization. Various strategies have been devised for the stable and efficient expression of heterologous antigens by attenuated S. enterica strains, but these methods often require complex manipulations. Use of phage lambda Red recombinase has recently been devised for gene replacements in Escherichia coli and S. enterica after introduction of PCR products. Based on this method, we have developed an approach that allows the integration of recombinant expression cassettes for heterologous antigens in a single step. The recombinant construct is integrated into the chromosome and is devoid of any selective marker such as antibiotic resistance. We observed the stable expression of model antigens without selective pressure. In addition, the method allows the simultaneous generation of attenuating mutations by gene deletions. The novel "knock-in" approach allows the rapid and efficient construction of recombinant Salmonella strains as vaccine carriers.

Animals↗
PubMed2006

Genetic analysis of simian immunodeficiency virus expressed in milk and selectively transmitted through breastfeeding.

To develop effective intervention strategies that prevent breast milk transmission of human immunodeficiency virus (HIV), we must understand the specific viral properties and mechanisms responsible for infant infection. We have used lactating rhesus macaques infected with a pathogenic simian immunodeficiency virus (SIV) stock to analyze the viral genotypes expressed in plasma and milk throughout the disease course and to identify those variants ultimately transmitted to infants through breastfeeding. In these studies we observed mother-to-infant transmission of SIV/Delta(B670) by eight females during the chronic phase of disease, and we analyzed by heteroduplex tracking assays and sequence analysis the distribution and fluctuations in viral genotypes expressed. Each female expressed multiple V1 envelope genotypes in milk near the time of transmission, while a single genotype was found in each of the infants. Variants transmitted to infants were not expressed throughout the maternal disease course but were only detected near the time of transmission. The emergence of the transmitted genotype in the dam typically occurred in plasma before milk and was coincident with increased milk viral loads. Transmitted genotypes tended to be longer and more glycosylated and had a less negative charge over the V1 region compared to viral genotypes expressed in milk but not transmitted. These observations demonstrate that specific viral genotypes are selectively transmitted to infants through breastfeeding and support the hypothesis that transmission occurs as genotypes adapt for efficient expression in milk.

Amino Acid Sequence↗
PubMed1990

Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies.

Animals↗
PubMed2002

Intratumoral low-volume jet-injection for efficient nonviral gene transfer.

Jet-injection has become an applicable technology among other established nonviral delivery systems, such as particle bombardment or in vivo electroporation. The low-volume jet injector employed in this study uses compressed air to inject solutions of 1.5-10 microL containing naked DNA into the desired tissue. The novel design of this prototype makes multiple jet-injections possible. Therefore, repeated jet-injections into one target tissue can be performed easily. This jet-injector hand-held system was used for the direct in vivo gene transfer of plasmid DNA into tumors to achieve efficient expression of reporter genes (beta-galactosidase, green fluorescent protein [GFP]) and of therapeutic genes (TNF-alpha) in different tumor models. The study presented here revealed the key parameters of efficient in vivo jet-injection (jet-injection volume, pressure, jet penetration, DNA stability) to define the optimal conditions for a jet-injection-aided nonviral gene therapy.

Animals↗
PubMed1992

A modified vector for the controlled high-level overproduction of staphylococcal protein A fusion proteins in the periplasm of Escherichia coli.

A vector encoding the Staphylococcal protein A was modified by cloning the spa gene, including its signal peptide-encoding sequence, downstream of the translation initiation sites of the phage lambda cro gene and under the control of the temperature-inducible phage lambda pR promoter. The expression from this construct was studied using the Escherichia coli phoA gene as a reporter gene after fusion to the spa gene. Determination of alkaline phosphatase activity, 1 h after temperature induction of expression at 42 degrees C, revealed an 800-fold increase over host strain background level. The presence of the alternating selectable markers on the described vector, pHEMa153, which are essential for efficient oligonucleotide-directed construction of mutations by the gapped duplex DNA method, allows the construction of recombinant and mutated forms of Staphylococcal protein A fusion proteins and efficient expression of spa gene fusions without changing the vector system.

Amino Acid Sequence↗
PubMed2004

[Construction and expression of the fusion vector of His-tagged human ARPC2 gene].

OBJECTIVE: To construct the expression vector of His-ARPC2 fusion protein and obtain its expression and purification in E. coli. METHODS: ARPC2 cDNA codon region was amplified by PCR from human liver cDNA library and cloned into pET-14b vector following the routine procedures. After identification by enzyme digestion, PCR and sequencing, the positive clones were transformed into BL21 (DE3) competent cells, and the expression of His-ARPC2 fusion protein was induced with IPTG and further purified by Ni-NTA affinity chromatography. RESULTS: The constructed His-ARPC2 fusion protein vector was highly efficiently expressed in E. coli. With Ni-NTA affinity chromatography, a purified His fusion protein with relative molecular mass of approximately 36 000 was obtained. CONCLUSION: The expression vector of His-ARPC2 fusion protein is constructed, expressed and purified under non-denaturing conditions, which may significantly facilitate future study of the physiological functions of ARPC2 and characterization of its interaction proteins.

Actin-Related Protein 2↗