Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield.
In the majority of the methods for purifying and concentrating recombinant adenoviruses (rAds) the virus that is associated with the helper cells is harvested, while the virus that is present in the cell-culture medium is discarded. During routine propagation of adenovirus type-5 vectors at optimised conditions we noted that, on average, 47% of the total amount of virus is present in the culture medium. To recover and concentrate these rAds from the medium, we devised a method, which is based on ammonium sulphate ((NH4)2SO4) precipitation. At 40% (NH4)2SO4 saturation, 95 +/- 6% of the available virus precipitates from the medium, while the majority of the protein (85%) remains in solution. In contrast to adenovirus precipitation with polyethylene glycol, the (NH4)2SO4 precipitation technique allows collection of precipitated rAds by filtration. We demonstrate here that (NH4)2SO4 precipitation of rAds from cell-culture medium is a simple and fast technique that can be used in combination with standard virus isolation methods to increase the yields of rAds.
Schagen
F H
FH
Department of Molecular Cell Biology, Leiden University Medical Centre, The Netherlands.
Rademaker
H J
HJ
Rabelink
M J
MJ
van Ormondt
H
H
Fallaux
F J
FJ
van der Eb
A J
AJ
Hoeben
R C
RC
D000256:Adenoviridae / Q000302:isolation & purification*; D000645:Ammonium Sulfate; D000818:Animals; D002460:Cell Line; D003470:Culture Media; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000302:isolation & purification*; D008156:Luciferases / Q000235:genetics
D003470:Culture Media; D008156:Luciferases; D000645:Ammonium Sulfate