RRC ID 2271
Author Ugai H, Yamasaki T, Hirose M, Inabe K, Kujime Y, Terashima M, Liu B, Tang H, Zhao M, Murata T, Kimura M, Pan J, Obata Y, Hamada H, Yokoyama KK.
Title Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration.
Journal Biochem. Biophys. Res. Commun.
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.
Volume 331(4)
Pages 1053-60
Published 2005-6-17
DOI 10.1016/j.bbrc.2005.03.227
PII S0006-291X(05)00752-7
PMID 15882984
MeSH Adenoviridae / isolation & purification* Adenoviridae / physiology Blotting, Western Electrophoresis, Polyacrylamide Gel Filtration / methods* HeLa Cells Humans Ultracentrifugation / methods* Virus Replication
IF 2.705
Times Cited 18
WOS Category BIOPHYSICS BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material anonymous