title: Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration.
abstract: Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 degrees C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35x10(10) pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.
authors.affiliation: Gene Engineering Division, BioResource Center, Tsukuba Institute, RIKEN (The Institute of Physical and Chemical Research), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
authors.forename: Hideyo
authors.initials: H
authors.lastname: Ugai
authors.forename: Takahito
authors.initials: T
authors.lastname: Yamasaki
authors.forename: Megumi
authors.initials: M
authors.lastname: Hirose
authors.forename: Kumiko
authors.initials: K
authors.lastname: Inabe
authors.forename: Yukari
authors.initials: Y
authors.lastname: Kujime
authors.forename: Miho
authors.initials: M
authors.lastname: Terashima
authors.forename: Bingbing
authors.initials: B
authors.lastname: Liu
authors.forename: Hong
authors.initials: H
authors.lastname: Tang
authors.forename: Mujun
authors.initials: M
authors.lastname: Zhao
authors.forename: Takehide
authors.initials: T
authors.lastname: Murata
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
authors.forename: Jianzhi
authors.initials: J
authors.lastname: Pan
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Obata
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Hamada
authors.forename: Kazunari K
authors.initials: KK
authors.lastname: Yokoyama
mesh_terms: D000256:Adenoviridae / Q000302:isolation & purification* / Q000502:physiology; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D014461:Ultracentrifugation / Q000379:methods*; D014779:Virus Replication