Inducible Expression Vector Research Articles

Mechanisms of Replication-Deficient Vaccinia Virus/T7 RNA Polymerase Hybrid Expression: Effect of T7 RNA Polymerase Levels and α-Amanitin

Components of the eukaryotic vaccinia virus/T7 RNA polymerase hybrid expression system were assessed using recombinant and nonrecombinant forms of modified vaccinia Ankara (MVA), a replication-deficient vaccinia virus strain. Recombinant MVA virus expressing T7 RNA polymerase (Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995).Virology210, 202–205) stimulated high levels of expression from a T7 promoter–chloramphenicol acetyltransferase (CAT) reporter. Most, but not all, of the virally induced expression was T7 RNA polymerase and T7 promoter dependent, with no viral enhancement of translation of T7 transcripts. The efficacy of supplying T7 RNA polymerase expression from nonviral sources was evaluated using a self-amplifying T7 RNA polymerase autogene or an inducible T7 RNA polymerase expression vector. The latter modes yielded CAT activity dependent on T7 RNA polymerase expression; however, expression required viral factors independent of T7 RNA polymerase and did not reach that attained using the recombinant virus. In further experiments, MVA-induced T7 RNA polymerase expression was upregulated by α-amanitin, an inhibitor of eukaryotic polymerases. This indicates that MVA/T7 RNA polymerase hybrid expression may be rendered still more efficient by ameliorating transcriptional interference due to an α-amanitin-sensitive eukaryotic factor(s).

Bioactive steroids as contaminants of the common carbon source galactose

Most inducible expression vectors for the budding yeast Saccharomyces cerevisiae are based on galactose-inducible promoters. Yeast has been increasingly used to study vertebrate steroid receptors because of its powerful genetics. In principle, both regulatory systems are compatible and can be combined in the same strain. However, we found that commercial galactose can be contaminated by bioactive estrogen and progesterone at concentrations that are sufficient to fully activate their cognate receptors. Since steroids can elicit biological responses in pathogenic fungi and possibly other microorganisms, such contaminants in a commonly used fermentable carbon source may need to be screened for.

Construction of a Sorbitol-Based Vector for Expression of Heterologous Proteins in Saccharomyces cerevisiae

A new inducible yeast expression vector, pXS7, was constructed by using the promoter and terminator sequences from the Saccharomyces cerevisiae SOR1 gene, which codes for the sorbitol dehydrogenase protein. We cloned the coding sequence of the Saccharomyces YEF3 gene in this vector and demonstrated an increase in YEF3 protein levels when cells were grown in the presence of the sugar sorbitol.

Bioactive steroids as contaminants of the common carbon source galactose.

Most inducible expression vectors for the budding yeast Saccharomyces cerevisiae are based on galactose-inducible promoters. Yeast has been increasingly used to study vertebrate steroid receptors because of its powerful genetics. In principle, both regulatory systems are compatible and can be combined in the same strain. However, we found that commercial galactose can be contaminated by bioactive estrogen and progesterone at concentrations that are sufficient to fully activate their cognate receptors. Since steroids can elicit biological responses in pathogenic fungi and possibly other microorganisms, such contaminants in a commonly used fermentable carbon source may need to be screened for.

A series of broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines

A series of shuttle vectors have been constructed that allow expression of heterologous proteins in either dipteran or lepidopteran insect cell lines. Constitutive expression in a broad range of host cells is mediated by the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus ( OpMNPV) immediate-early 2 ( ie2) promoter. Alternatively, if inducible expression is required, for example to express cytotoxic proteins, a vector has been constructed that uses the Drosophila metallothionein (Mtn) promoter for metal-inducible protein expression in dipteran cell lines. A chimeric synthetic bacterial- OpMNPV ie promoter-Zeocin ™ resistance gene cassette has been included to facilitate cloning in E. coli as well as the generation of stably transformed insect cell lines. The utility of the system is demonstrated by the constitutive and inducible expression of the highly processed glycosylphosphatidylinositol-anchored glycoprotein, human melanotransferrin, in transformed insect cell lines.

Enhancement of chemosensitivity toward anticancer drugs by high expression of caspase-1 in NIH 3T3 cells.

It has been well documented that caspase-1 (interleukin-1beta-converting enzyme, ICE) and its related cysteine proteinases such as caspase-3 (CPP32, apopain) and caspase-2 (ICH-1L) play important roles in apoptosis. In the present study, these genes were inserted into an inducible eukaryotic expression vector, pMSG, and transfected into NIH 3T3 mouse fibroblasts. The expression of caspases-1 and -3 was effectively induced by treatment with dexamethasone (Dex). The expression of caspase-2 was elevated in the transfected cells without treatment with Dex but was not further stimulated by Dex. High expression of these proteases alone induced neither apoptosis-like cell death nor any morphological change. However, the expression of caspase-1 but not of caspase-2 or -3 enhanced chemosensitivity toward cytotoxic anticancer drugs such as aclarubicin, epirubicin, adriamycin, nimustine and ifosfamide. It is thus concluded that caspase-1 mediates cytotoxic effects of these drugs.

Suppression of phospholipase C beta, gamma, and delta families alters cell growth and phosphatidylinositol 4,5-bisphosphate levels.

Phosphatidylinositol-specific phospholipase C (PLC) activity reflects a summation of the activities of three families, beta, gamma, and delta, each of which is regulated differently. In order to understand the contribution of each family to cell proliferation signaling, expression of each family was suppressed by use of an inducible expression vector for antisense PLC sequences in a single cell line, FTO-2B rat hepatocytes. Activation of second messengers of PLC [diacylglycerol (DAG) and inositol 1,4,5-tris(phosphate) (IP3)] was dramatically reduced, providing a strategy for probing the consequences of PLC deficiency on cell function. Importantly, while one PLC family was suppressed, the other PLCs actively responded to specific stimuli, suggesting parallel and independent signaling pathways for each PLC family in FTO-2B cells. Selective suppression of each PLC family altered cell growth markedly and differentially. The rank order for suppression of cell growth by loss of a PLC family was gamma > delta > beta. Exploration of down-stream growth regulators revealed that loss of beta and gamma, but not delta, families was associated with markedly reduced basal ras and protein kinase C activity. Moreover, suppression of each of the three PLC families caused remarkably reduced basal and stimulated MAP kinase activities. Interestingly, cellular levels of PIP2 were increased and dramatically correlated with growth inhibition rate in the clones with suppressed PLC activity, suggesting that PIP2 itself can serve as a second messenger of cell growth regulation.

The major antigenic protein of infectious bursal disease virus, VP2, is an apoptotic inducer.

Infectious bursal disease virus (IBDV) is the causative agent of an economically important poultry disease. Vaccinia virus recombinants expressing the IBDV mature structural capsid proteins VP2 and VP3 were generated by using vectors for inducible gene expression. Characterization of these recombinant viruses demonstrated that expression of VP2 leads to induction of apoptosis in a variety of mammalian cell lines. Transfection of cell cultures with a expression vector containing the VP2 coding region under the control of the immediate-early promoter-enhancer region of human cytomegalovirus also triggers programmed cell death. The apoptotic effect of VP2 is efficiently counteracted by coexpression of the proto-oncogene bcl-2. The results presented demonstrate that VP2 is a bona fide apoptotic inducer. Evaluation of the significance of this finding for the virus life cycle must await further research.

Employment of the mdr1 promoter for the chemotherapy-inducible expression of therapeutic genes in cancer gene therapy.

Numerous approaches in gene therapy of human cancers are focused on the establishment of cell type specific or inducible expression vectors allowing the targeted and regulated expression of therapeutic genes. Various conditionally active vectors have been created carrying promoters responding to certain factors or therapeutic modalities (eg hormones, irradiation). The promoter of the multidrug resistance gene (mdr1) harbors such responsive elements and two of these elements have been related to drug responsiveness. In earlier studies we and others have characterized the mdr1 drug responsive-element in CAT reporter assays demonstrating its inducibility by MDR-associated drugs. To exploit this property, we linked the mdr1 promoter sequence to the human tumor necrosis factor alpha (TNF) cDNA in a retroviral vector and transduced the vector into human mammary and colon carcinoma cell lines. These cells were treated with various mdr1-associated drugs to induce TNF expression in vitro. We have shown that the mdr1 promoter-driven TNF expression is drug-inducible and that this induction is drug concentration and time dependent. The studies demonstrate the feasibility of the novel vector system for a chemotherapy-inducible expression of a chemosensitizing cytokine that is successful at enhancing cytotoxicity of drugs in cancer therapy.

Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1α promoter and lac operator

We have constructed an inducible high-level expression vector, pEF-LAC. pEF-LAC has a modified human polypeptide chain elongation factor 1α (EF-1α) promoter containing three lactose operator sequences. Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC. In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1α promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR). Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl β- d-thiogalactopyranoside (IPTG). Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner. These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.

High-level expression of the chemically synthesized gene for microbial transglutaminase from Streptoverticillium in Escherichia coli.

We developed a novel approach for the high-level production of a microbial transglutaminase (TGase) from Streptoverticillium in E. coli. The direct expression of the TGase gene in E. coli cells did not cause overproduction, probably due to the harmful influence of TGase activity, which introduces covalent crosslinks between proteins. Therefore, we fused the chemically synthesized TGase gene coding for the entire 331 amino acid residues at the amino terminus to a bacteriophage T7 gene 10 leader peptide (260 amino acids) using an inducible expression vector. The TGase gene was expressed as inclusion bodies in the E. coli cytoplasm. Restoring 15 amino acid residues upstream of the amino terminus of the mature TGase by a two-step deletion of the fusion sequence facilitated solubilization and subsequent proteolytic cleavage, thus releasing mature TGase. Although the mature form had less TGase activity than native TGase, because of the poor refolding rate, these results suggest that this system is suitable for the efficient production of TGase.

A transgenic mouse model with an inducible skin blistering disease phenotype.

One of the current limitations of gene transfer protocols involving mammalian genomes is the lack of spatial and temporal control over the desired gene manipulation. Starting from a human keratin gene showing a complex regulation as a template, we identified regulatory sequences that confer inducible gene expression in a subpopulation of keratinocytes in stratified epithelia of adult transgenic mice. We used this cassette to produce transgenic mice with an inducible skin blistering phenotype mimicking a form of epidermolytic hyperkeratosis, a keratin gene disorder. Upon induction by topical application of a phorbol ester, the mutant keratin transgene product accumulates in the differentiating layers of epidermis, leading to keratinocyte lysis after application of mechanical trauma. This mouse model will allow for a better understanding of the complex relationship between keratin mutation, keratinocyte cytoarchitecture, and hypersensitivity to trauma. The development of an inducible expression vector showing an exquisite cellular specificity has important implications for manipulating genes in a spatially and temporally controlled fashion in transgenic mice, and for the design of gene therapy strategies using skin as a tissue source for the controlled delivery of foreign substances.

Insertion of metal-responsive elements increases the inducibility of the mouse mammary tumor virus promoter

The promoter of the mouse mammary tumor virus (MMTV) is commonly used in inducible mammalian expression vectors. We have modified this promoter by inserting metal-responsive elements ( MRE) at different sites. Surprisingly, MMTV promoters containing MRE 5′ with respect to the glucocorticoid responsive elements (GRE), were not induced by metal ions. However, in the simultaneous presence of dexamethasone and metal ions, the modified promoters displayed a significantly increased transcriptional strength [up to sevenfold more than wild-type (wt) MMTV] in transiently transfected cells and permanent cell lines. This increment in the inducible expression was achieved without a concomitant increase in the basal level of expression. These modified MMTV promoters will offer the opportunity to develop tightly regulated inducible mammalian expression systems that are significantly more potent than the ones based on the wt MMTV promoter.

Nucleotide sequences of three distinct cDNA clones coding for the rat class I heavy chain RT1n antigen.

We isolated and sequenced three different RT1n cDNA clones that each encode the entire major histocompatibility complex (MHC) class I heavy chain protein, including the 39 untranslated region (UTR) and the poly-A tail. The mRNA was isolated from Concanavalin A-stimulated (Pharmacia, Piscataway, NJ) splenocytes of BN (RT1n) rats (Harlan Sprague-Dawley, Indianapolis, IN). The cDNA library was synthesized from BN mRNA using an reverse transcription (RT)-polymerase chain reaction (PCR)-based cloning method (Wang et al. 1996). Using previously described upstream and downstream primers in a PCR-based method (Wang et al. 1996) we isolated three RT1n clones. To assure the accuracy of each RT1n clone, we individually sequenced the cDNA samples from triplicate PCR reactions as well as from triplicate TA-cloning reactions. Multiple RT1n clones for each cDNA were then sequenced in both directions to eliminate any possibility of random PCR mutations. Although we performed sequencing of multiple clones, we cannot completely exclude the possibility of PCR artifact. As previously suggested (Joly et al. 1995), undesirable PCR reactions such as polymerase “jumping” between different sequences are possible, especially at high concentrations of templates. We found that our three RT1n sequences are similar to rat class I MHC sequences available in the GenBank, including two RT1n sequences (Joly et al. 1995; X90375, X90376; Fig. 1). Interestingly, our clones 4 and 12 are identical in the 39 UTR and at the 39 half of the coding region. Furthermore, the sequence of our clone 12 (U50448) is similar to the partial cDNA sequence of RT1n (X90375) with the exception of two nucleotides (GC) at positions 564 and 565, which differ from those (CG) presented in their sequence. This difference in our amino acid sequence produced Trp and Leu amino acids at positions 191 and 192, instead of Cys and Val amino acids as in the RT1n (X90375) sequence. Our three cDNAs were subcloned into pMAMneo (CloneTech, Palo Alto, CA), an inducible eukaryotic expression vector, and transfected into rat BUF (RT1b) hepatoma cells. Neomycin (Life Technologies; Grand Island, NY)-resistant transfectants (0.5 × 106) stimulated with 1 μg dexamethasome (DEX; Sigma, St. Louis, MO) for 6 h were tested for cell-membrane expression of class I MHC molecules by FACS analysis with an FITC-conjugated mouse anti-rat class I MHC-specific monoclonal antibody (OX18; Pharmingen, San Diago, CA). Both non-induced and DEXinduced normal BUF cells expressed almost identical baseline amounts of class I RT1.Ab molecules (Fig. 2 A, B). Similarly, DEX-stimulated control BUF cells that had been transfected with pMAMneo alone (without RT1n cDNA) expressed a similar number of class I MHC molecules as non-transfected controls (Fig. 2 C). In contrast, following DEX induction, BUF hepatoma cells transfected with any of three RT1n clones displayed increased expression of membrane-bound class I MHC molecules (Fig. 2 D, E, F). Thus, our results document that all three transfected RT1n genes may be expressed on the cell surface, when under control of a DEX-inducible promoter.

Overexpression of pokeweed antiviral protein in Escherichia coli: purification, characterization and comparison with native sources

Pokeweed antiviral proteins (PAP, PAP-II, PAP-S) represent a family of ribosome inactivating proteins (RIPs) isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). They not only inhibit the mechanical transmission of plant viruses but also inactivate both eukaryotic and prokaryotic protein synthesis. One of them, PAP, was expressed with the help of the highly inducible prokaryotic expression vector pOPE 90 in Escherichia coli and the biological activity of the recombinant PAP (rPAP) was compared with the PAP purified from spring leaves of Phytolacca americana. After extraction and solubilization, rPAP was highly purified by anion and cation exchange chromatography. Immunoblotting with antibodies revealed identical immunological reactivity between recombinant or native PAP. In a rabbit reticulocyte translation system, rPAP was more efficient than native PAP to inhibit protein synthesis. The values of IC 50 from native PAP and recombinant PAP were determined as 5.10 −11 M and 5.10 −12 M, respectively. No difference was observed when the toxicity of native and rPAP was tested on established cell lines. PAP can be expressed in equivalent active form in Escherichia coli and its properties were thus similar to those of the native protein. Post-translational modifications of PAP in pokeweed did not seem to alter its ability to inhibit protein synthesis.

Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions.

Increased autocrine production of insulin-like growth factor II (IGF-II) alters serum sensitivity of MCF-7 human breast cancer cell proliferation.

Regulation of the growth of breast cancer cells is the result of a complex interaction between steroid hormones and growth factors, and in particular of oestrogen and insulin-like growth factors (IGF). Alteration of any one mitogenic component can affect the cell response to other pathways. Previous work has shown that increased autocrine production of IGF-II from a transfected inducible expression vector can result in reduced oestrogen sensitivity of growth of MCF-7 human breast cancer cells. This report describes alterations to non-oestrogen regulated pathways of cell growth following enhanced IGF-II expression in these transfected MI7 cells. Serum sensitivity of cell growth in the absence of oestrogen was found to differ between MI7 and untransfected MCF-7 cells, in that growth of MI7 but not MCF-7 cells was strongly inhibited by high serum levels. Increased serum had no effect on levels of IGF-II mRNA, IGFIR, IGFBP4 mRNA, or IGFBP secreted in MI7 cells. However, growth inhibition by serum in MI7 cells could be overcome by increasing levels of IGF-II in the serum or by removal of IGFBP onto polycarbonate membranes. Thus, the growth inhibition by serum in MI7 cells is concluded to result from the increased levels of IGFBP added with higher serum. This would support an inhibitory role for IGFBP on growth of breast cancer cells when cell growth is being driven by IGF pathways in the absence of oestrogen, and would suggest that cellular sensitivity to such factors can depend on levels of endogenous IGF production.

Large-Scale Purification and Characterization of Recombinant Fibroblast Growth Factor-Saporin Mitotoxin

In order to produce sufficient quantities of fibroblast growth factor-saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large-scale expression, purification, and characterization of the recombinant molecule. The fusion gene encoding rFGF-2-SAP was cloned into the inducible pET 11a expression vector and transformed intoEscherichia colistrain BL21 (DE3). The transformants were grown using a fed-batch fermentation until theA600reached 85. At this stage, induction of the expression of the fusion protein led to the production of ∼2.2 mg/liter perA600unit. The soluble mitotoxin was purified to homogeneity from cell lysates via expanded bed adsorption chromatography followed by cation-exchange, heparin-affinity, and size-exclusion chromatography. Purified rFGF-2-SAP contained less than 0.5 EU/mg of endotoxin, as determined by gel clot analyses. The highly purified rFGF-2-SAP retained the toxin's ability to inhibit protein synthesis as measured in a cell-free system and was cytotoxic to a number of normal and neoplastic cell lines bearing FGF receptors. Binding studies establish that the fusion protein exerts its effects via the FGF high-affinity receptor.

DEMONSTRATION OF ALKALINE PHOSPHATASE PARTICIPATION IN THE MINERALIZATION OF OSTEOBLASTS BY ANTISENSE RNA APPROACH

MC3T3-E1 cells grown with ascorbic acid express sequentially osteoblastic marker proteins such as alkaline phosphatase (ALPase) and then form a mineralized extracellular matrix (ECM) as a consequence of osteoblastic differentiation. To explore the functional roles of ALPase in the process of osteoblastic maturation, an inducible expression vector for antisense ALPase RNA was constructed and stably transfected into MC3T3-E1 cells. The expression of antisense ALPase RNA in the differentiated MC3T3-E1 transfectants reduced markedly the ALPase activity, which resulted in a significant decrease in the deposition of minerals upon prolonged culture. These findings demonstrated directly that ALPase participated in the mineralizationof ECM.

Cloning, sequencing and functional expression of a DNA encoding pig cytosolic malate dehydrogenase: purification and characterization of the recombinant enzyme

Using the polymerase chain reaction, DNA encoding cytosolic malate dehydrogenase (cMDH) has been cloned from a pig heart cDNA library. Large amounts of the enzyme (30 mg per litre of original culture) have been produced in Escherichia coli using an inducible expression vector (pKK223-3) in which the 5′-non-coding region of the gene was replaced with the tac promoter. The complete nucleotide sequence of the DNA is reported for the first time. The recombinant cMDH purified was shown to be identical to the native enzyme according to: chromatographic behaviour, isoelectric point, N-terminal amino acid sequence, and physicochemical and catalytic properties.