Purpose: We developed a prokaryotic expression system to express the major capsid protein of Polyomavirus, VP1. Furthermore, we investigated the transport of single stranded (ss) and double stranded (ds) DNA, mediated through VP1 as drug delivery system into mouse fibroblasts.
Methods: To study DNA delivery we used two kinds of DNA, a ssDNA fragment (19mer) and dsDNA (plasmid pEGFPN1, 4.7 kb or a FITC-labelled dsDNA fragment, 1.8 kb).
Results: The uptake of VP1 capsoids loaded with FITC-labelled oligodeoxynucleotides (FODNs) was observed. VP1 pentamers loaded with condensates of dendrimer/dsDNA fragments (FITC-labelled) resulted in significantly higher fluorescence signal in the cytoplasm of NIH 3T3 cells in comparison to control experiments without VP1. Additionally, VP1 capsoids loaded with plasmid pEGFPN1 without dendrimers resulted in an approximately 10 fold higher transfection rate in comparison to blank DNA controls.
Conclusions: Our results demonstrated the potential of VP1 capsoids as DNA delivery system. EGFP expression was significantly enhanced when plasmid DNA was delivered via VP1 capsoids, compared to control experiments with naked DNA.
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http://dx.doi.org/10.1023/a:1026497411053 | DOI Listing |
Biotechnol J
December 2006
november AG, Erlangen, Germany.
Highly immunogenic capsomers (pentamers) and virus-like particles (VLPs) were generated through insertion of foreign B cell epitopes into the surface-exposed loops of the VP1 protein of murine polyomavirus and via heterologous expression of the recombinant fusion proteins in E. coli. Usually, complex proteins like the keyhole limpet hemocyanin (KLH) act as standard carrier devices for the display of such immunogenic peptides after chemical linkage.
View Article and Find Full Text PDFJ Biol Chem
June 2004
Responsif GmbH, Erlangen, Germany.
Efficient encapsulation of foreign molecules like proteins and low molecular weight drugs into polyoma virus-like particles (capsoids) was achieved by the development of an anchoring technique based upon the specific interaction of the inner core protein VP2 with VP1 pentamers. A stretch of 49 amino acids of VP2 served as an anchor molecule, either expressed as a fusion protein with green fluorescent protein (GFP) or covalently linked to methotrexate (MTX). The loaded capsoids showed regular morphology and stability for several months.
View Article and Find Full Text PDFPharm Res
September 2000
Institute for Pharmaceutical Technology, Biocenter-Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.
Purpose: We developed a prokaryotic expression system to express the major capsid protein of Polyomavirus, VP1. Furthermore, we investigated the transport of single stranded (ss) and double stranded (ds) DNA, mediated through VP1 as drug delivery system into mouse fibroblasts.
Methods: To study DNA delivery we used two kinds of DNA, a ssDNA fragment (19mer) and dsDNA (plasmid pEGFPN1, 4.
Biotechnol Appl Biochem
February 1999
Institute for Pharmaceutical Technology, University of Frankfurt, Biozentrum, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany.
The drug delivery system described here is based on the properties of the capsoid or capsid-like structure resulting from the assembly of polyoma virus capsid protein VP1 expressed in Escherichia coli. The capsid protein VP1 was expressed as a fusion protein with a completely removable N-terminal His6 affinity tag. The pentameric morphology of the recombinant VP1 protein was confirmed by electron microscopy after affinity chromatography and factor Xa cleavage under conditions of low ionic strength.
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