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Biomedical subjects

Reingard Grabherr

Publications and source records attributed to Reingard Grabherr.

12 recordsLinked to original sources
PubMed2006

Improving baculovirus transduction of mammalian cells by surface display of a RGD-motif.

An RGD-containing peptide, comprising 23 amino acids from the foot-and-mouth disease virus (FMDV) VP1 protein was engineered into the envelope of Autographa californica nuclear polyhedrosis virus surface (AcNPV) using two different display strategies. The RGD-motif is a well-described tripeptide, that by binding to cell surface integrins facilitates virus entry into cells. This epitope was displayed, either by directly modifying the native major envelope protein gp64 of AcNPV, or by incorporating a second, modified version of gp64 onto the virus surface. Transduction efficiencies of four mammalian cell lines were compared by detecting the expression of the reporter gene green fluorescent protein (gfp), delivered by the baculovirus genome. Our results showed that insertion of the RGD-peptide into the envelope protein gp64 leads to enhanced specific uptake of baculoviral particles in mammalian cells, only when a combination of wild-type and mutant gp64 was present on the viral surface. Whenever the RGD-peptide was directly inserted into the native gp64, the overall amount of gp64 envelope protein was diminished, leading to decreased viral uptake.

Amino Acid Motifs↗
PubMed2006

RGD motifs on the surface of baculovirus enhance transduction of human lung carcinoma cells.

Baculovirus vectors have been shown to enter a variety of mammalian cell lines and gene transfer with wild-type baculovirus (WT) has been demonstrated both in vitro and in vivo. Different protein motifs have been displayed on the viral surface to serve as ligands for cell-specific receptor molecules. We have generated recombinant baculovirus vectors displaying an RGD-motif, recognized by alphaV integrin, on the viral surface. The RGD motifs within the C-terminus of coxsackie virus A9 and human parechovirus 1 VP1 proteins were fused to the N-terminus of the major envelope glycoprotein, gp64, of Autographa californica multiple nucleopolyhedrovirus. The recombinant RGD-presenting viruses bound more efficiently to the surface of human lung carcinoma cells (A549), known to contain alphaV integrins, as compared to WT baculovirus. In addition, the binding pattern of the RGD-displaying baculovirus showed extensive clustering. This most likely represents clustering of the integrin molecules on the cell surface, induced by binding of the RGD-displaying baculovirus. Finally, the transduction efficiency of an RGD-representing virus increased by almost three-fold as monitored by light emission measurements. In conclusion, these results suggest that the RGD-motif is functional on the surface of baculovirus and thereby these tropism-modified viruses bind more efficiently as well as enhance the transduction efficiency of human cancer cells expressing alphaV integrins.

Animals↗
PubMed2006

Using ColE1-derived RNA I for suppression of a bacterially encoded gene: implication for a novel plasmid addiction system.

The use of plasmid DNA for gene therapeutical purposes is a novel technology with advantages and drawbacks. One of the required improvements is to avoid antibiotic resistance genes or other additional sequences for selection within the plasmid. Here, we describe an alternative approach to equip a ColE1 plasmid with a regulatory function within the cell, which could be used for selection of plasmid carrying cells. No additional sequences are required, since the mechanism is based on RNA/RNA antisense interaction involving the naturally occurring RNA I derived from the plasmid's origin of replication. The plasmid replicational regulatory network was linked to the transcriptional regulatory network of an engineered target gene, present on the bacterial chromosome. Thus, gene suppression of a reporter could be achieved by mere presence of the ColE1-type plasmid pBR322. Proof of this concept was shown in shaker-flask experiments and fed-batch fermentation processes. The strategy of regulating gene expression by plasmid replication implicates a novel strategy for plasmid selection, as the gene to be suppressed could be toxic or growth hampering, providing advantage to plasmid carrying host cells.

Bacterial Proteins↗
PubMed2004

Influence of promoter choice and trichostatin A treatment on expression of baculovirus delivered genes in mammalian cells.

The expression of recombinant proteins following transduction of CHO cells with recombinant baculoviruses containing a mammalian expression cassette with the CMV-promoter is enhanced by the addition of trichostatin A (TSA), a specific histone deacetylase inhibitor. To further investigate the effect of TSA treatment on protein production following BacMam transduction, viruses containing various viral promoters (SV40, CMV, and RSV) and one cellular promoter (human ubiquitin C) were compared with regard to expression level of a gfp-luciferase fusion protein following transduction of CHO, COS-1, and HEK293 cells. The overall effect on expression appears to be cell specific, indicating that different mechanisms are active within different cell lines. Further, COS cells transfected with naked viral DNA, plasmids, and baculovirus particles were compared in regard to TSA treatment. The increase in reporter gene expression observed following BacMam transduction and TSA treatment were greater than those for transfection of either naked viral DNA or plasmid DNA.

Animals↗
PubMed2004

Improved display of synthetic IgG-binding domains on the baculovirus surface.

Improved display of foreign protein moieties in combination with beneficial alteration of the viral surface properties should be of value for targeted and enhanced gene delivery. Here, we describe a vector based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV) displaying synthetic IgG-binding domains (ZZ) of protein A fused to the transmembrane anchor of vesicular stomatitis virus (VSV) G protein. This display vector was equipped with a GFP/EGFP expression cassette enabling fluorescent detection in both insect and mammalian cells. The virus construct displayed the biologically active fusion protein efficiently and showed increased binding capacity to IgG. As the display is carried out using a membrane anchor of foreign origin, gp64 is left intact for virus entry, which may increase gene expression in the transduced mammalian cells. In addition, the viral vector can be targeted to any desired cell type via binding of ZZ domains when an appropriate IgG antibody is available.

Animals↗
PubMed2003

Generation of recombinant influenza virus using baculovirus delivery vector.

A recombinant baculovirus vector containing mammalian cell-active promoters and transcription terminators was used to deliver a mutated influenza NS gene into Vero cells. In addition to the influenza NS gene, the baculovirus contained a reporter gene expression cassette (Green fluorescent protein, GFP), allowing to monitor the Vero cell transduction efficiency. More than 90% of Vero cells were expressing GFP 24-48 h post transduction. After infecting baculovirus transduced cells with influenza helper virus, progeny of attenuated influenza virus carrying the recombinant NS gene could be selected. Baculovirus delivery was highly reproducible and efficient in Vero cells. This new method for influenza gene delivery could contribute to influenza virus research and vaccine development.

Animals↗
PubMed2003

Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery.

Recombinant baculoviruses have been extensively used as vectors for abundant expression of a large variety of foreign proteins in insect cell cultures. The appeal of the system lies essentially in easy cloning techniques and virus propagation combined with the eukaryotic post-translational modification machinery of the insect cell. Recently, a novel molecular biology tool was established by the development of baculovirus surface display, using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a versatile system in molecular biology. Surface display strategies play an important role, as they may be used to enhance the efficiency and specificity of viral binding and entry to mammalian cells. In addition, baculovirus surface display vectors have been engineered to contain mammalian promoter elements designed for gene delivery both in vitro and in vivo. Moreover, baculovirus capsid display has recently been developed; this holds promise for intracellular targeting of the viral capsid and subsequent cytosolic delivery of desired protein moieties. Finally, the viruses can accommodate large insertions of foreign DNA and replicate only in insect cells. Together, these are attributes that are very likely to make them important tools in functional genomics and proteomics.

Animals↗
PubMed2002

Stabilizing plasmid copy number to improve recombinant protein production.

The key objective for recombinant protein production in bacteria is the maximum exploitation of the cell factory's potential, whereby often strong expression vectors are used to increase product yield. If the metabolic load caused by recombinant expression exceeds the host's capacity, the system exhausts itself, resulting in a loss of protein yield. Excessive plasmid replication is observed after inducing recombinant gene expression, which greatly contributes to metabolic overload of the host cell. The transcriptional and translational machineries are extremely overstrained. By abolishing sequence homology between ColE1 RNA I/RNA II and tRNAs, we were able to restore the plasmid's replication control mechanisms and to keep the plasmid copy number constant throughout the culture process, thereby prolonging metabolic activity and productivity of the bacterial expression system. Because the bacterial host cell is not being exploited beyond its tolerable potential with this method, the constancy of the plasmid copy number level throughout the whole period of the bioprocess provides novel strategies for bioprocess optimization.

Bacterial Proteins↗
PubMed2002

Impact of targeted vector design on Co/E1 plasmid replication.

The demands for recombinant proteins, in addition to plasmid DNA, for therapeutic use are steadily increasing. Bacterial fermentation processes have long been and still are the major tool for production of these molecules. The key objective of process optimization is to attain a high yield of the required quality, which is determined, to a large extent, by plasmid replication rates, metabolic capacity and the properties of the specific gene construct. When high copy number plasmids are used, the metabolic capacity of the host cell is often overstrained and efficient protein production is impaired. The plasmid copy number is the key parameter in the exploitation of the host cell, and can be maximized by optimal control of the flux ratios between biosynthesis of host cell proteins and recombinant proteins.

Bacterial Proteins↗
PubMed2002

Age-related alterations in the protein expression profile of C57BL/6J mouse pituitaries.

The aim of our study was to monitor the protein expression profile in pituitary glands of healthy C57BL/6J mice during aging. Pituitary glands of 4-week old (immature), 3-month old (mature), and >25-month old mice were analysed by proteomic tools such as two-dimensional electrophoresis and N-terminal micro-sequencing. A change was detected in the expression of growth hormone after sexual maturation. Our particular interest, however, was directed against up-regulated proteins in the old pituitary glands, which are proposed to be involved in the process of neuroendocrine aging. Among these proteins, the expression of glutathione-S-transferase (GST) and apolipoprotein A-1 were increased in old pituitaries. Furthermore, ubiquitin carboxyl-terminal hydrolase (UCH-L1) was significantly up-regulated in senescent C57BL/6J mouse pituitaries. Since only the rat homologue was known, we isolated and analysed the mouse UCH-L1 sequence. Since GST is involved in antioxidative defence and UCH-L1 is part of the ubiquitin/proteasome system, which is responsible for the removal of damaged proteins, these results suggest increased oxidative burden and an increased activity of the ubiquitin system.

Aging↗
PubMed2002

Altering the surface properties of baculovirus Autographa californica NPV by insertional mutagenesis of the envelope protein gp64.

The envelope protein gp64 of the baculovirus Autographa californica nuclear polyhedrosis virus is essential for viral entry into insect cells, as the glycoprotein both mediates pH-dependent membrane fusion and binds to host cell receptors. Surface modification of baculovirus particles by genetic engineering of gp64 has been demonstrated by various strategies and thus has become an important and powerful tool in molecular biology. To improve further the presentation of peptides on the surface of baculovirus particles, several insertion sites within the gp64 envelope protein were selected by their theoretical maximum surface probability and investigated for efficient peptide presentation. The ELDKWA peptide of the gp41 of HIV-1, specific for the human mAb 2F5, was inserted into 17 different positions of the glycoprotein gp64. Propagation of viruses was successful in 13 cases, mutagenesis at four positions did not result in production of intact virus particles. Western blotting, FACS analysis and ELISA were used for characterization of the different binding properties of the mutants. Insertion of this peptide into the native envelope protein resulted in high avidity display on the surface of baculovirus particles. This approach offers the possibility of effective modification of surface properties in regard to host range specificity and antigen display.

Amino Acid Sequence↗