Large-scale production, purification and crystallization of wild-type adeno-associated virus-2.
Adeno-associated virus-2 (AAV-2) has long been recognized as a potential vector for human gene therapy. Although much progress has been made in the molecular virology of AAV-2, structural studies of AAV-2 have been hampered by the low efficiency of virus production in culture, the low purity of preparations, and the low solubility of pure virus particles in solution. Methods of larger scale AAV-2 production have been developed through adaptation to suspension culture and re-optimization of the times of infection and transfection with respect to particle production. The methods allow the purification of 10mg ( approximately 10(15) particles) of AAV-2 per preparation at approximately 99% purity as judged by SDS-PAGE. This was sufficient for the screening of conditions for the formation of diffraction-grade crystals, ultimately leading to an atomic structure for AAV-2.
Xie
Qing
Q
Kasha Laboratory of Molecular Biophysics, Florida State University, FL, USA.
Hare
Joan
J
Turnigan
Joy
J
Chapman
Michael S
MS
D003460:Crystallization; D000229:Dependovirus / Q000737:chemistry / Q000254:growth & development* / Q000302:isolation & purification* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry; D014773:Virology / Q000379:methods*; D014776:Virus Cultivation; D014779:Virus Replication
D014764:Viral Proteins