Search results for "Expression vector"
showing 10 items of 31 documents
Functional analysis of a rare HBV deletion mutant in chronically infected children.
2003
Liver damage caused by chronic hepatitis B virus (HBV) infection may be enhanced through the selection of deleted HBV preS mutants by intracellular accumulation of viral proteins and subsequent cell death. However, the prevalence and impact of such mutants on the clinical course of infection have not yet been studied in children. Serum samples from 60 children (mean age 9.8 y) were investigated by means of PCR and direct sequencing of the entire preS region. Only one patient (1.5%) was found with a mixed HBV population of a deletion spanning 183 nucleotides and wild-type sequences. This mutation alters the HBV large-surface protein and removes the small-surface promoter. To clarify the sign…
Advanced Strategies for Food-Grade Protein Production: A New E. coli/Lactic Acid Bacteria Shuttle Vector for Improved Cloning and Food-Grade Expressi…
2019
Food-grade production of recombinant proteins in Gram-positive bacteria, especially in LAB (i.e., Lactococcus, Lactobacillus, and Streptococcus), is of great interest in the areas of recombinant enzyme production, industrial food fermentation, gene and metabolic engineering, as well as antigen delivery for oral vaccination. Food-grade expression relies on hosts generally considered as safe organisms and on clone selection not dependent on antibiotic markers, which limit the overall DNA manipulation workflow, as it can be carried out only in the expression host and not in E. coli. Moreover, many commercial expression vectors lack useful elements for protein purification. We constructed a &ld…
Baculovirus Display: A Multifunctional Technology for Gene Delivery and Eukaryotic Library Development
2006
For over a decade, phage display has proven to be of immense value, allowing selection of a large variety of genes with novel functions from diverse libraries. However, the folding and modification requirements of complex proteins place a severe constraint on the type of protein that can be successfully displayed using this strategy, a restriction that could be resolved by similarly engineering a eukaryotic virus for display purposes. The quite recently established eukaryotic molecular biology tool, the baculovirus display vector system (BDVS), allows combination of genotype with phenotype and thereby enables presentation of eukaryotic proteins on the viral envelope or capsid. Data have sho…
Yeast vectors for the integration/expression of any sequence at theTYR1 locus
2007
We have constructed new yeast vectors for targeted integration and conditional expression of any sequence at the Saccharomyces cerevisiae TYR1 locus which becomes disrupted. We show that vector integration is not neutral, causing prototrophy for tyrosine and auxotrophy for the vector's selectable marker (uracil or leucine, depending on the vector used). This feature allows a double screening of transformed yeast cells, improving the identification of colonies with the desired chromosomal structure. The GAL10 gene promoter has been added to drive conditional expression of cloned sequences. Using these vectors, chromosomal structure verification of recombinant clones is no longer necessary, s…
A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica.
1996
A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library. The cDNA library was constructed in the expression vector lambda gt 11, with the RNA isolated from actively growing mycelial cells. The deduced amino acid sequence shows that the encoded protein contains an N-terminal hydrophobic signal peptide. We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein. Northern hybridization identified YWP1 transcript only when Y. lipolytica was growing in the mycelial form. The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is …
Is tubulin the sole antigen recognized by a putative anti-bursicon antibody?
1999
Abstract A 56-kDa polypeptide suspected to be the tanning hormone `bursicon' was analyzed using the monoclonal antibody (mAb) 01C10 of Song and Ma. We studied the beetle Tenebrio molitor, for which data on bursicon have been recently published. After purification by two-dimensional gel electrophoresis of brain proteins, the immunoreactive 56-kDa polypeptide was trypsinated and microsequenced. The obtained sequences revealed a high homology with α- and β-tubulins. In a complementary study, immunoreactive clones were isolated, using the 01C10 mAb, from a library in expression vector obtained from Drosophila melanogaster head cDNAs. Again, the isolated clones were found, after cDNA sequencing,…
Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin.
2003
Strong cell-type-specific promoters are basic tools in gene therapy allowing for novel applications and focused strategies by transcriptionally targeting gene expression to selected cells. In immunotherapy, dendritic cells (DC) are of central importance, since they represent the principal inducers of immune responses. Here we describe isolation and use of the promoter of the murine actin-bundling protein fascin to target transcriptionally gene expression to cutaneous DC. Using the reporter gene enhanced green fluorescent protein (EGFP), we demonstrate that the fascin promoter mediates a strong antigen expression that is restricted to mature DC. DNA vaccination with antigen-encoding expressi…
Transcriptional Regulation of Human CYP3A4 Basal Expression by CCAAT Enhancer-Binding Protein α and Hepatocyte Nuclear Factor-3γ
2003
Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more than 50% of currently used therapeutic drugs, yet the mechanisms that control CYP3A4 basal expression in liver are poorly understood. Several putative binding sites for CCAAT/enhancer-binding protein (C/EBP) and hepatic nuclear factor 3 (HNF-3) were found by computer analysis in CYP3A4 promoter. The use of reporter gene assays, electrophoretic mobility shift assays, and site-directed mutagenesis revealed that one proximal and two distal C/EBP alpha binding sites are essential sites for the trans-activation of CYP3A4 promoter. No trans-activation was found in similar reporter gene experiments with a HNF-3 gamma expression vec…
The Mu1 transposable element of maize contains two promoter signals recognized by the Escherichia coli RNA polymerase.
1990
The galactokinase (GalK) expression plasmid vector system pKO-1 has been used to screen for promoter elements in the maize transposable element Mu1 that function in Escherichia coli. Two transcriptional start points, named S1 and S2, were identified, which are located in the two direct repeats of the transposable element. This paper demonstrates that sequence elements exist in a plant transposable element which function as prokaryotic promotors.
Stearoyl-CoA desaturase 1 coding sequences abd antisense RNA affect lipid secretion in transfected chicken LMH hepatoma cells
2000
Hepatic stearoyl CoA desaturase (SCD) activity in chickens from a fat line is higher than that of chickens from a lean line and correlates with plasma triacylglycerol concentrations. Furthermore, in these lines, the hepatic SCD1 mRNA level is positively correlated with the adipose tissue weight. To analyze the contribution of the SCD1 gene in the regulation of adiposity in the early stages of triacylglycerol secretion, SCD1 coding sequence and antisense RNA expression vectors were transfected in LMH cells. After selection, these cells were analyzed with regard to SCD1 expression and lipid secretion. The amounts of secreted triacylglycerols and phospholipids were shown to be higher in LMH ce…