Extracellular self-assembly of virus-like particles from secreted recombinant polyoma virus major coat protein.
Mouse polyoma virus major coat protein (VP1) expressed from a recombinant baculovirus is efficiently transported to infected cell nuclei and assembles into protein nanospheres morphologically similar to natural capsids. The nanospheres readily combine with plasmid DNA to form a hybrid gene therapy agent known as virus-like particles (VLPs). To facilitate large-scale production of VLPs free from cellular contaminants, the use of stable Drosophila cell lines expressing either wild-type protein, or VP1 tagged with a secretion signal for targeting to the extracellular medium, was investigated. Both wild-type and tagged VP1 expressed at 2-4 mg VP1/litre of culture. As expected, the wild-type protein self-assembled into VLPs. The tagged VP1 was efficiently secreted to the extracellular medium but was also glycosylated, unlike wild-type VP1. Despite this fact, a small fraction of the recombinant secreted protein assembled into VLP-like structures that had altered disulphide bonding, but were still biologically active. These results demonstrate the considerable tolerance in the nanosphere assembly to structural (i.e. aberrant glycosylation) and environmental (i.e. extracellular medium vs. nuclear milieu) changes. Thus, with modifications to improve nanosphere assembly, the secretion method could be adapted to large-scale preparation of VLPs, providing significant advantages over current methods of production of the vector.
Ng
J
J
MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK.
Koechlin
O
O
Ramalho
M
M
Raman
D
D
Krauzewicz
N
N
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004330:Drosophila; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D054118:Nanospheres / Q000737:chemistry; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011120:Polyomavirus / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry*; D014415:Tunicamycin / Q000494:pharmacology
D004220:Disulfides; D011994:Recombinant Proteins; D014415:Tunicamycin