Purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation.
Sucrose equilibrium density ultracentrifugation remains the most widely used technique for retrovirus purification. However, purified virus preparations obtained by this routine method usually contain considerable amounts of contaminating cell membrane vesicles. In addition, sucrose solutions are highly viscous and hyperosmotic which jeopardizes the integrity and functionality of the retrovirus particle. In order to overcome these limitations, an alternative purification technique using rate zonal ultracentrifugation and iodixanol as gradient medium was developed. Recombinant retrovirus particles were produced by 293-GPG packaging cells grown in suspension in the presence of 10% FBS. Concentrated supernatants were purified by rate zonal sedimentation on a 10-30% continuous iodixanol gradient. Virus particles were recovered intact and active from the central fractions of the gradient. By using this strategy, high levels of purification were achieved, with no evident contamination with cell membrane vesicles as indicated by subtilisin treatment studies. The level of purity of the retrovirus preparation is over 95% as shown by SDS-PAGE analysis and size-exclusion chromatography. Purified particles appear homogenous in size and morphology according to negative stain electron microscopy. In addition, large amounts of defective retrovirus particles produced by 293-GPG packaging cells can be separated from functional retrovirus particles using this purification strategy.
Segura
María de las Mercedes
MM
Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montreal, Qué., Canada H4P 2R2. amine.kamen@cnrc-nrc.gc.ca
Garnier
Alain
A
Kamen
Amine
A
D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002850:Chromatography, Gel; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000494:pharmacology; D005374:Filtration; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000302:isolation & purification; D010316:Particle Size; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D016622:Silver Staining; D018139:Simplexvirus / Q000201:enzymology; D020860:Subtilisin / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014283:Triiodobenzoic Acids / Q000494:pharmacology; D014461:Ultracentrifugation; D014771:Virion / Q000737:chemistry* / Q000302:isolation & purification* / Q000648:ultrastructure
D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D014283:Triiodobenzoic Acids; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D004720:Endonucleases; D020860:Subtilisin; C044834:iodixanol