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At least 1,009 records · Page 56Linked to original sources
PubMed2005

Environmentally controlled invasion of cancer cells by engineered bacteria.

Bacteria can sense their environment, distinguish between cell types, and deliver proteins to eukaryotic cells. Here, we engineer the interaction between bacteria and cancer cells to depend on heterologous environmental signals. We have characterized invasin from Yersinia pseudotuburculosis as an output module that enables Escherichia coli to invade cancer-derived cells, including HeLa, HepG2, and U2OS lines. To environmentally restrict invasion, we placed this module under the control of heterologous sensors. With the Vibrio fischeri lux quorum sensing circuit, the hypoxia-responsive fdhF promoter, or the arabinose-inducible araBAD promoter, the bacteria invade cells at densities greater than 10(8)bacteria/ml, after growth in an anaerobic growth chamber or in the presence of 0.02% arabinose, respectively. In the process, we developed a technique to tune the linkage between a sensor and output gene using ribosome binding site libraries and genetic selection. This approach could be used to engineer bacteria to sense the microenvironment of a tumor and respond by invading cancerous cells and releasing a cytotoxic agent.

Adhesins, Bacterial↗
PubMed2006

A semi-automated large-scale process for the production of recombinant tagged proteins in the Baculovirus expression system.

The efficient preparation of recombinant proteins at the lab-scale level is essential for drug discovery, in particular for structural biology, protein interaction studies and drug screening. The Baculovirus insect-cell expression system is one of the most widely applied and highly successful systems for production of recombinant functional proteins. However, the use of eukaryotic cells as host organisms and the multi-step protocol required for the generation of sufficient virus and protein has limited its adaptation to industrialized high-throughput operation. We have developed an integrated large-scale process for continuous and partially automated protein production in the Baculovirus system. The instrumental platform includes parallel insect-cell fermentation in 10L BioWave reactors, cell harvesting and lysis by tangential flow filtration (TFF) using two custom-made filtration units and automated purification by multi-dimensional chromatography. The use of disposable materials (bags, filters and tubing), automated cleaning cycles and column regeneration, prevent any cross-contamination between runs. The preparation of the clear cell lysate by sequential TFF takes less than 2 h and represents considerable time saving compared to standard cell harvesting and lysis by sonication and ultra-centrifugation. The process has been validated with 41 His-tagged proteins with molecular weights ranging from 20 to 160 kDa. These proteins represented several families, and included 23 members of the deubiquitinating enzyme (DUB) family. Each down-stream unit can process four proteins in less than 24 h with final yields between 1 and 100 mg, and purities between 50 and 95%.

Animals↗
PubMed2006

The complete sequence and segregational stability analysis of a new cryptic plasmid pIGWZ12 from a clinical strain of Escherichia coli.

A new cryptic plasmid from a multi-resistant, multi-plasmid clinical strain of Escherichia coli has been isolated. The sequence of the 4072-base-pair pIGWZ12 (GenBank Accession No. DQ311641) was determined and analyzed. Two open-reading frames that code for proteins involved in plasmid mobilization and initiation of replication were identified. The putative origin of replication possesses all characteristic features of the theta mechanism for replicating plasmids. pIGWZ12 is stably maintained without selective pressure in bacterial cultures (for up to 80 generations), making it a good candidate for engineering a new cloning vector.

Amino Acid Sequence↗
PubMed2004

Taming plastids for a green future.

Plant genetic engineering will probably contribute to the required continued increase in agricultural productivity during the coming decades, and moreover, plants can potentially provide inexpensive production platforms for pharmaceuticals and nutraceuticals. With the advent of technologies for altering the genetic information inside chloroplasts, a new attractive target for genetic engineering has become available to biotechnologists. Potential advantages over conventional nuclear transformation include high transgene expression levels and increased biosafety because of maternal organelle inheritance in most crops. This review summarizes the state of the art in chloroplast genetic engineering and describes how reverse genetics approaches enhance our understanding of photosynthesis and other important chloroplast functions. Furthermore, promising strategies by which chloroplast genetic engineering might contribute to the successful modification of plant metabolism are discussed.

Chloroplasts↗
PubMed2005

Making the most of affinity tags.

Proteins do not naturally lend themselves to high-throughput analysis because of their diverse physiochemical properties. Consequently, affinity tags have become indispensable tools for structural and functional proteomics initiatives. Although originally developed to facilitate the detection and purification of recombinant proteins, in recent years it has become clear that affinity tags can have a positive impact on the yield, solubility and even the folding of their fusion partners. However, no single affinity tag is optimal with respect to all of these parameters; each has its strengths and weaknesses. Therefore, combinatorial tagging might be the only way to harness the full potential of affinity tags in a high-throughput setting.

Animals↗
PubMed2004

Construction and evaluation of genetically engineered replication-defective porcine reproductive and respiratory syndrome virus vaccine candidates.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an emerging pathogen causing significant economic losses in the swine industry worldwide. Two novel gene-deleted viruses were constructed and evaluated as vaccine candidates. Using the full-length infectious cDNA clone of North American PRRS isolate P129, the ORF2 and ORF4 genes (which encoded minor structural glycoproteins GP2a/2b and GP4, respectively) were individually deleted from the viral genome. Both deletion mutants were non-viable in MARC-145 cells and porcine alveolar macrophages, indicating that both genes are essential for virus replication. To rescue the replication-defective PRRSV, two complementing cell lines, MARC-2000 and MARC-400, were established to stably express the PRRSV GP2 and GP4 proteins, respectively. These cells were able to complement the deleted gene function of PRRSV in trans and supported production of the replication-defective DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses. Both DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses were propagated for 40-50 generations in the corresponding complementing cells and remained replication-defective in MARC-145 cells. To examine the immunogenic potential of the replication-defective PRRSV as vaccine candidates, four groups of pigs, 20 pigs per group, were immunized twice with DeltaORF2-PRRSV or DeltaORF4-PRRSV and challenged with the homologous virulent virus at 3 weeks post-immunization. In spite of the fact one group showed significant reduction in virus load, we could not demonstrate improvement from clinical diseases in this vaccination/challenge study. However, we did show that the cDNA clone of PRRSV can be a useful tool to genetically engineer PRRSV vaccine candidates and to study pathogenesis and viral gene functions.

Animals↗
PubMed2005

Construction and applications of yellow fever virus replicons.

Subgenomic replicons of yellow fever virus (YFV) were constructed to allow expression of heterologous reporter genes in a replication-dependent manner. Expression of the antibiotic resistance gene neomycin phosphotransferase II (Neo) from one of these YFV replicons allowed selection of a stable population of cells (BHK-REP cells) in which the YFV replicon persistently replicated. BHK-REP cells were successfully used to trans-complement replication-defective YFV replicons harboring large internal deletions within either the NS1 or NS3 proteins. Although replicons with large deletions in either NS1 or NS3 were trans-complemented in BHK-REP, replicons that contained deletions of NS3 were trans-complemented at lower levels. In addition, replicons that retained the N-terminal protease domain of NS3 in cis were trans-complemented with higher efficiency than replicons in which both the protease and helicase domains of NS3 were deleted. To study packaging of YFV replicons, Sindbis replicons were constructed that expressed the YFV structural proteins in trans. Using these Sindbis replicons, both replication-competent and trans-complemented, replication-defective YFV replicons could be packaged into pseudo-infectious particles (PIPs). Although these results eliminate a potential role of either NS1 or full-length NS3 in cis for packaging and assembly of the flavivirus virion, they do not preclude the possibility that these proteins may act in trans during these processes.

Cell Line↗
PubMed2005

Generation of serum-stabilized retroviruses: reduction of alpha1,3gal-epitope synthesis in a murine NIH3T3-derived packaging cell line by expression of chimeric glycosyltransferases.

Retroviral vectors released from mouse-derived packaging cell lines are inactivated in human sera by naturally occurring antibodies due to the recognition of Galalpha1,3Galbeta1,4GlcNAc (alphagal-epitope) decorated surface proteins. In this study, an extensive analysis of the glycosylation potential of NIH3T3-derived PA317 packaging cells using combined MALDI/TOF-MS and HPAE-PAD reveals that 34% of the N-glycan moiety represents alphagal-epitope containing structures. Stable expression of glycosyltransferases and transport signal chimeras has been demonstrated to represent an efficient tool to alter cell- and species-specific glycosylation (Grabenhorst and Conradt, 1999. J. Biol. Chem. 274, 36107-36116). In order to reduce alphagal-epitope synthesis selected chimeric glycosyltransferases were constructed by fusing Golgi-signal sequences for compartment-specific localization with the catalytic domain of alpha2,3-sialyltransferase (ST3). Stable expression of these constructs in these cells resulted in a significant reduced alphagal-epitope synthesis, and moreover, a release of retroviral vectors showing an up to 3.5-fold increase in serum stability. Thus, our results suggest that the stably transfected cells stably transfected with chimeric glycosyltransferases compete efficiently with endogenous alpha1,3-galactosyltransferase. This approach allows favored glycodesign and we anticipate the applicability of such improved retroviral vectors produced by glycosylation engineered host cells for in vivo gene therapy and, furthermore, suggest the therapeutic benefit of this technology for xenotransplantation.

Animals↗
PubMed2006

6-hydroxy-nicotine-inducible multilevel transgene control in mammalian cells.

The precise control of transgene expression is essential for biopharmaceutical manufacturing, gene therapy and tissue engineering. We have designed a novel conditional transcription technology, which enables reversible induction, repression and adjustment of desired transgene expression using the clinically inert 6-hydroxy-nicotine (6HNic). The 6-hydroxy-nicotine oxidase (6HNO) repressor (HdnoR), which manages nicotine metabolism in Arthrobacter nicotinovorans pAO1 by binding to a specific operator of the 6-hydroxy-nicotine oxidase (O(NIC)), was fused to the Krueppel-associated box protein of the human kox-1 gene (KRAB) to create a synthetic 6HNic-dependent transsilencer (NS) that controls chimeric mammalian promoters, which are assembled by cloning tandem O(NIC) operators 3' of a constitutive promoter. In the absence of 6HNic, NS binds to O(NIC) and silences the constitutive promoter, which otherwise drives high-level transgene expression when the NS-O(NIC) interaction stops in the presence of 6HNic. Generic NICE(ON) technology was compatible with a variety of constitutive viral and mammalian housekeeping promoters, each of which enabled specific induced, repressed, adjusted and reversible transgene expression profiles in Chinese hamster ovary (CHO-K1), baby hamster kidney (BHK-21) as well as in human fibrosarcoma (HT-1080) cells. NICE(ON) also proved successful in controlling multicistronic expression units for coordinated transcription of up to three transgenes and in the fine-tuning of transcription-translation networks, in which RNA polymerase II- and III-dependent promoters, engineered for 6HNic responsiveness, drove expression of siRNAs that triggered specific transgene knockdown. NICE(ON) represents a robust and versatile technology for the precise tuning of transgene expression in mammalian cells.

Animals↗
PubMed2005

Construction of tumor-specific toxins using ubiquitin fusion technique.

The use of cytotoxic agents to eliminate cancer cells is limited because of their nonselective toxicity and unwanted side effects. One of the strategies to overcome these limitations is to use latent prodrugs that become toxic in situ after being enzymatically activated in target cells. In this work we describe a method for producing tumor-specific toxins by using a ubiquitin fusion technique. The method is illustrated by the production of recombinant toxins by in-frame fusion of ubiquitin to saporin, a toxin from the plant Saponaria officinalis. Ubiquitin-fused toxins were rapidly degraded via the ubiquitin-proteasome system, significantly reducing their nonspecific toxicity. The insertion of the protease-cleavage sequence between ubiquitin and saporin led to the removal of ubiquitin by the protease and resulted in protease-dependent stabilization of the toxin. We engineered toxins that can be stabilized by specific proteases such as deubiquitinating enzymes and prostate-specific antigen (PSA). Both constructs were activated in vitro and in cultured cells by the appropriate enzyme. Processing by the protease resulted in a greater than 10-fold increase in the toxicity of these constructs. Importantly, the PSA-cleavable toxin was able to kill specifically the PSA-producing prostate cancer cells. The ubiquitin fusion technique is thus a versatile and reliable method for obtaining selective cytotoxic agents and can easily be adapted for different kinds of toxins and activating proteases.

Cell Line, Tumor↗
PubMed2001

High-yield expression of a viral peptide vaccine in transgenic plants.

A high-yield production of a peptide vaccine in transgenic plants is described here. A 21-mer peptide, which confers protection to dogs against challenge with virulent canine parvovirus, has been expressed in transgenic plants as an amino-terminal translational fusion with the GUS gene. Transformants were selected on the basis of their GUS activities, showing expression levels of the recombinant protein up to 3% of the total leaf soluble protein, a production yield comparable to that obtained with the same epitope expressed by chimeric plant viruses. The immunogenicity of the plant-derived peptide was demonstrated in mice immunized either intraperitoneally or orally with transgenic plant extracts, providing the suitability of the GUS fusions approach for low-cost production of peptide vaccines.

Administration, Oral↗
PubMed2002

Production of a recombinant antimicrobial peptide in transgenic plants using a modified VMA intein expression system.

Tobacco plants were engineered to express SMAP-29, a mammalian antimicrobial peptide of innate immunity, as fusion protein with modified vacuolar membrane ATPase intein. The peptide was purified taking advantage of the intein-mediated self-cleaving mechanism. SMAP-29 was immunologically detected in the chromatographic eluate and appeared tightly bound to copurified plant proteins. Electrophoretic separation under disaggregating conditions indicated that the recombinant peptide was cleaved off by intein at the expected site and an overlay gel assay demonstrated that the peptide retained antimicrobial activity. These results indicate that a modified intein expression system can be used to produce pharmaceutical peptides in transgenic plants.

Blood Proteins↗
PubMed2003

Preproinsulin mRNA engineering and its application to the regulation of insulin secretion from human hepatomas.

Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include non-beta cells genetically engineered to secrete insulin in response to physiologic cues; responsiveness can be introduced at the transcriptional level to regulate preproinsulin (PPI) mRNA biosynthesis. However, these cells exhibit sluggish secretion dynamics, which is not appropriate for achieving euglycemia in higher animals and, eventually, humans. In this work, we have engineered the PPI mRNA so as to destabilize it through nonsense-mediated mRNA decay (NMD). When expressed under transcriptional regulation in HepG2 hepatomas, the engineered PPI mRNA level and of the insulin secretion rate declined faster upon switching off transcription, compared to the one-copy non-engineered control. Our work provides a simple and straightforward method to improve the dynamics of transcriptionally regulated insulin secretion, which can be a useful tool in developing cell-based therapies for IDD.

Base Sequence↗