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We had earlier overproduced the transcription activator protein C of bacteriophage Mu in a phage-T7 expression system. Although we achieved a high level of overproduction, the expression was not consistent. This could be due to the leaky expression of T7 RNA polymerase in the uninduced state. Introduction of pLysS, a plasmid encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase, resulted...
We have constructed phagemid and phage-based vectors which can be used for both direct (T/A) and ligation-independent cloning (LIC) of PCR products for surface display of encoded peptides/proteins fused with the gIII protein of the filamentous bacteriophages M13 and fd-tet. The vectors harbour a DNA cassette consisting of the lacZα fragment inserted between the + 2 and + 3 codons of gIIIp. The lacZα...
We have used the methylotrophic yeast, Pichia pastoris, to express the open reading frame 3 (ORF3) of the hepatitis E virus (HEV). The ORF3 gene codes for a 123-amino-acid protein that contains highly immunodominant epitopes and is a potentially useful diagnostic and immunoprophylactic antigen. The expressed protein showed positive on immunoblots probed against antibodies raised in rabbit and infected...
Promoter function of the putative polyhedrin-encoding gene (polh) of Spodoptera litura nuclear polyhedrosis virus (SlMNPV) was determined by transferring it to the Autographa californica nuclear polyhedrosis virus (AcMNPV) through the AcNPV polh based vector, pVL1393. Three transfer vectors pCBT2, pCBT3 and pCBT4 were constructed by substituting the promoter and the neighbouring sequences of AcNPV...
The baculovirus expression vector is a eukaryotic DNA viral vector for the cloning and expression of foreign genes in cultured lepidopteran insect cells and insects. It has become an important tool for the large-scale production of recombinant proteins for a variety of applications including the structure-function analysis of genes and their gene products. We have developed a number of baculovirus...
The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based...
Chimeric genes containing the genomic fragments of hepatitis B virus (HBV) coding for middle (M) and major/small (S) surface proteins were constructed and expressed in transgenic potato plants under the control of the cauliflower mosaic virus 35S promoter. Enzyme-linked immunosorbent assay (ELISA) revealed the presence of HBV surface antigen (HBsAg) determinants in extracts from transgenic plants...
DNA-dependent RNA polymerases (RNApol) are Zn 2+ metalloproteins where the Zn 2+ ion plays both catalytic and structural roles. Although the ubiquitous presence of Zn 2+ with the RNApol from eukaryotes had already been established, the exact stoichiometry of Zn 2+ ion(s) per mole enzyme is not well documented, and its role in enzymatic function...
Adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus, is gaining attention as a vector for its potential use in human gene therapy. However, few studies have examined the safety and the efficacy of this vector system in vivo. We report here that recombinant AAV vectors, when directly injected intravenously in mice, accumulated predominantly in liver cells, suggesting that AAV may possess...
Alphavirus vectors have become widely used in basic research to study the structure and function of proteins and for protein production purposes. Development of a variety of vectors has made it possible to deliver foreign sequences as naked RNA or DNA, or as suicide virus particles produced using helper vector strategies. Preliminary reports also suggest that these vectors may be useful for in vivo...
A 637-bp fragment, corresponding to the p24 human immunodeficiency virus (HIV) core protein from the gag ORF, was PCR amplified from DNA isolated from peripheral blood mononuclear lymphocytes (PBML) of an asymptomatic HIV-1 seropositive human subject from Bombay and cloned into PCRScript SK(+). The nucleotide sequence revealed highest homology (98.6%) with the consensus sequence of the HIV-1 B subtype...
We have already reported that human fibroblast 34-kDa hyaluronic acid-binding protein (HABP) is identical with P32, the protein co-purified with splicing factor SF-2 [Deb and Datta (1996) J. Biol. Chem. 271, 2206-2212]. Data search further revealed that it has 92% sequence homology with a murine protein YL2 which interacts with HIV1 Rev. In this paper we have successfully demonstrated that HIV1...
Perfusion of liver with plasmid DNA-lipofectin complexes via the portal vein results in efficient accumulation of the vector in hepatocytes. Such hepatocytes, when administered intraperitoneally into a hepatectomized rat, repopulate the liver and express the transgene efficiently. This procedure obviates the need for large-scale hepatocyte culture for ex vivo gene transfer. Further, intraperitoneal...
Baculovirus expression vectors were constructed, which contained gfp as a reporter gene. Substitutions in the amino acid sequences were carried out to produce two mutant forms of GFP. One of these mutants produces blue color when excited by ultraviolet (UV) light. The other mutant produces a yellow color that can be visualized in regular daylight. The gene of interest cloned in-frame with the...
Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP 3 ), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca 2+ channel, the IP 3 receptor (IP 3 R), which is therefore a central component of this signaling cascade. Here we describe...
From the onset of gene technology yeasts have been among the most commonly used host cells for the production of heterologous proteins. At the beginning of this new development the attention in molecular biology and biotechnology focused on the use of the best characterized species, Saccharomyces cerevisiae, leading to an increasing number of production systems for recombinant compounds. In recent...
The nucleotide (nt) sequence of the 3 end of the yeast HIS3 mRNA was determined by PCR amplification of the 3 end. Analysis of 28 individual clones revealed that at least 13 distinct polyadenylation sites are present. The sites of polyadenylation are extremely heterogeneous and do not show any obvious similarity other than that they occur after pyrimidine residues in most cases. Most mutants...
Cancer vaccines are based on the concept that tumors express novel antigens and thus differ from their normal tissue counterparts. Such putative tumor-specific antigens should be recognizable by the immune system. However, malignant cells are of self origin and only poorly immunogenic, which limits their capability to induce an anticancer immune response. To overcome this problem, tumor cells have...
The baculovirus expression vector system has emerged as the system of choice for the expression of a number of heterologous genes of both prokaryotic and eukaryotic origin. This system utilizes the baculovirus very late, hyperactive polyhedrin and p10 promoters to drive the transcription of foreign genes. Regulation of transcription from these promoters is presently not well understood even though...
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