Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration.
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.
Ugai
Hideyo
H
Gene Engineering Division, BioResource Center, Tsukuba Institute, RIKEN (The Institute of Physical and Chemical Research), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
Yamasaki
Takahito
T
Hirose
Megumi
M
Inabe
Kumiko
K
Kujime
Yukari
Y
Terashima
Miho
M
Liu
Bingbing
B
Tang
Hong
H
Zhao
Mujun
M
Murata
Takehide
T
Kimura
Makoto
M
Pan
Jianzhi
J
Obata
Yuichi
Y
Hamada
Hirofumi
H
Yokoyama
Kazunari K
KK
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