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Biomedical subjects

R Grabherr

Publications and source records attributed to R Grabherr.

12 recordsLinked to original sources
PubMed2006

Optimization of baculovirus transduction on FreeStyle293 cells for the generation of influenza B/Lee/40.

Recombinant baculovirus expression vectors derived from the Autographa californica nuclear polyhedrosis virus can serve as efficient gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types and are able to produce multisubunit particles such as viruses or virus like particles. In this study, we constructed eight recombinant baculoviruses each containing one of the influenza B/Lee/40 virus genes in a bidirectional expression cassette for simultaneous mRNA and viral RNA transcription. Baculoviruses were transduced into FreeStyle293 in combination with the specific histone deacetylase inhibitor trichostatin A (TSA). Cotransduction conditions were optimized with a set of five baculoviruses (influenza B/Lee/40 PB1, PB2, PA, and NP and the control construct NCR-NS-minus-sense orientated encoding green fluorescent protein [rGFP]), which led to GFP expression in each host cell transduced with all five constructs. Based on the optimization with five constructs, transduction with eight baculoviruses was performed at MOI 50 and 100 with high yield stocks and 1 microM TSA and led to successful rescue of infectious influenza B/Lee/40 viruses.

Animals↗
PubMed2001

Developments in the use of baculoviruses for the surface display of complex eukaryotic proteins.

The ability to couple genotype to phenotype has proven to be of immense value in systems such as phage display and has allowed genes encoding novel functions to be selected directly from complex libraries. However, the complexity of many eukaryotic proteins places a severe constraint on successful display in Escherichia coli. This restriction could be resolved if a eukaryotic virus could be similarly engineered for display purposes. Preliminary data have suggested that the baculovirus Autographa californica, a multiple nuclear polyhedrosis virus (AcMNPV) is a candidate for eukaryotic virus display because the insertion of peptides into the native virus coat protein, or the expression of foreign proteins as coat protein fusions, results in incorporation of the sequence of interest onto the surface of virus particles. A variety of strategies are currently under investigation to develop further the display capabilities of AcMNPV and to improve the complexity of library that might be accommodated. Several expression vectors for different forms of surface display have been developed and, coupled with improved recombination strategies, represent progress towards a refined tool for use in functional genomics and in vitro protein evolution.

Animals↗
PubMed2001

The baculovirus expression system as a tool for generating diversity by viral surface display.

It has become a major goal of molecular biologists, biochemists, and immunologists to be able to modulate the structure of proteins, in order to increase their antigenicity, alter their biological properties and/or explore their function. Based on the concept of bacterial phage display, by which proteins are being selected and analyzed in conjunction with their genetic information, eukaryotic systems have been investigated for their use in generating biomolecular diversity. The advantage of posttranslational modification and the possible harbouring of structural complex proteins has lead scientists to include eukaryotic systems in the wide field of molecular design. The ideal expression vectors for surface display are eukaryotic viruses, that allow large gene insertions, efficiently present foreign proteins on the particle surface, are easy to propagate and, if possible, not pathogenic to humans. By inserting peptides into a native virus coat protein or by expressing foreign proteins as coat protein fusion proteins or linked to specific anchor domains it becomes possible to display polypeptides of interest on the surface of replicating particles. A variety of different strategies are currently under investigation in order to utilize the baculovirus insect cell expression system for efficient display on the surface of virus particles as well as on the surface of virally infected insect cells. Increasing the transfection efficiency, optimizing cloning procedures, and establishing applicable selection methods have lead to the development of a powerful tool for drug screening and ligand screening.

Baculoviridae↗
PubMed1998

Baculovirus surface display: construction and screening of a eukaryotic epitope library.

The baculovirus expression system was utilized to serve as a tool for ligand selection, demonstrating the applicability of the system to the generation and screening of eukaryotic expression libraries. The HIV-1-gp41 epitope 'ELDKWA', specific for the neutralizing human mAb 2F5, was inserted into the antigenic site B of influenza virus hemagglutinin and expressed on the surface of baculovirus infected insect cells. In order to improve the antigenicity of the epitope within the hemagglutinin, and therefore enhance the specific binding of 2F5, we inserted three additional, random amino acids adjacent to the epitope. This pool of hemagglutinin genes was directly cloned into the baculovirus Ac-omega. To identify distinct proteins displayed on the cellular surface, we developed a screening protocol to select for specific binding capacity of individual viral clones. Using fluorescence activated cell sorting (FACS) we isolated a baculovirus clone displaying the epitope with markedly increased binding capacity out of a pool of 8000 variants in only one sorting step. Binding properties of the identified ligand were examined by FACS performing a competition assay.

Amino Acid Sequence↗
PubMed1997

Expression of foreign proteins on the surface of Autographa californica nuclear polyhedrosis virus.

Based on the method of direct cloning into the baculovirus genome by linearizing and re-ligation in presence of the target insert, we designed viral constructs that express foreign genes on the surface of baculovirus particles. We chose the glycosylated envelope protein gp41 of human immunodeficiency virus type 1 (HIV-1) as a model for displaying recombinant proteins on budded virus. The ectodomain of the envelope protein gp41 of HIV-1 was being fused to the entire baculovirus major coat protein gp64 (Ac-cops41) and to the membrane anchor sequence of gp64 (Acmars41). Two different promoters, the "very late" polyhedrin promoter (Ac-mars41) and the "early and late" gp64 promoter (Ac-promars41) were compared. The expression of gp41 in infected cells and its presence on viral particles was confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot and electron microscopy.

Animals↗
PubMed1996

Human interleukin-2 (IL-2) expressed by transfected mammalian cells.

cDNA for human interleukin-2 (IL-2) was cloned into the pRc/RSV vector for expression in animal cells. Baby hamster kidney (BHK-21) cells and Chinese hamster ovary (CHO) cells were transfected several times using calcium phosphate and electroporation methods with the construct pRc/RSV SIGIL2. Different transfection efficiencies were obtained. The biological test on CTLL-2 (mouse cytotoxic lymphocytes) showed that the kinetics of cell proliferation were different from those of rIL-2 (recombinant IL-2) expressed in bacteria and in BHK cells. When high concentrations of rIL-2 were applied, an inhibitory effect on CTLL-2 was observed when bacterial product was used, whilst rBHK interleukin caused no inhibition. Recombinant BHK IL2 induced a slower response of CTLL-2 cells at the beginning of the cultivation, however, prolonged activity was detected at the later stage of the experiment.

Animals↗
PubMed1994

Hairpin loop structure at the termini of the chlorella virus PBCV-1 genome.

The termini of the chlorella virus PBCV-1 330-kb dsDNA genome consist of 35-nucleotide-long, incompletely base-paired, covalently closed hairpin loops that exist in one of two forms. The two forms are complementary when the 35 nucleotide sequences are inverted with respect to one another (flip and flop). Each hairpin loop structure is followed by an identical 2221-bp inverted repeat sequence after which the DNA sequence diverges. The strategy for cloning the PBCV-1 DNA hairpin ends may be useful for cloning other hairpin termini.

Base Sequence↗
PubMed1993

Sequencing two DNA templates in five channels by digital compression.

By applying algebraic coding methods to the Sanger dideoxynucleotide procedure, DNA sequences of two templates can be determined simultaneously in only five reactions and data channels. A 5:2 data compression is accomplished by instantaneous source coding of nucleotide sequence pairs into one set of 5-bit block codes. A general algebraic expression, 2n-1 > or = 4f, describes conditions under which f DNA templates can be sequenced using n channels. Such compression sequencing is accurate and efficient, as demonstrated by manual 35S autoradiographic detection and automated on-line analysis using fluorescent-labeled primers. Symmetric 5:2 compression is especially useful when comparing two closely related sequences.

Automation↗
PubMed1992

DNA sequencing of four bases using three lanes.

A three-lane DNA sequencing strategy is described which is based on redundant binary coding principles from communications theory. Three-lane sequencing is an efficient, accurate, and flexible strategy, suitable for large-scale automated DNA sequencing. Communications theory and algebraic coding principles can also be applied to sequencing other informational macromolecules.

Base Composition↗
PubMed1992

The DNA polymerase gene from chlorella viruses PBCV-1 and NY-2A contains an intron with nuclear splicing sequences.

The deduced amino acid sequences of two eukaryotic chlorella virus (PBCV-1 and NY-2A) DNA polymerases are 90% identical and contain amino acid motifs typical of alpha-like (Family B) DNA polymerases. The open reading frames of both PBCV-1 and NY-2A DNA polymerases are interrupted by an identically located, small (101 or 86 nucleotides, respectively) intron that resembles eukaryotic nuclear-spliced messenger RNA introns. This discovery suggests that chlorella virus replication has a nuclear phase.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing the cytosine methyltransferase gene M. CviJI from Chlorella virus IL-3A.

The Chlorella virus IL-3A gene encoding the DNA methyltransferase M.CviJI, which methylates the internal cytosine in (G/A)GC(T/C/G) sequences, was cloned and expressed in Escherichia coli. The region containing the M.CviJI gene was sequenced and a single open reading frame of 1101 bp was identified that could code for a polypeptide of 367 amino acids with a predicted molecular weight of 41,864. M.CviJI contained regions of amino acids which were similar to bacterial cytosine methyltransferases. Eighteen other Chlorella viruses, of 36 tested, contained DNA sequences which hybridized to the M.CviJI gene; DNA from some, but not all, of these 18 viruses also contained 5-methylcytosine in (G/A)GC(T/C/G) sequences.

Amino Acid Sequence↗