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The zonal centrifuge applied to the purification of a low‐yield virus in human leukemia cells
Author(s) -
Toplin I.,
Boyden R.,
De Padova A.,
Brandt P.,
Sottong P.
Publication year - 1968
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260100510
Subject(s) - centrifugation , centrifuge , virus , density gradient , differential centrifugation , chromatography , sucrose , cell culture , lysis , chemistry , cell , sucrose gradient , biology , microbiology and biotechnology , virology , biochemistry , enzyme , physics , genetics , quantum mechanics , nuclear physics
The herpes‐type virus found in certain cell cultures derived from Burkitt's lymphoma, other human leukemias, and normal human leukocytes, was concentrated and partially purified by large‐volume density gradient centrifugation using zonal centrifuge systems. Using the Jiyoye (P‐3) cell line as a model, rate‐zonal runs on disrupted cell suspensions in sucrose gradients yielded concentrates with high virus particle counts when 10–15 ml of packed cells were processed per liter of gradient. Isolation and removal of cell nuclei or fluorocarbon treatment of cell sonicates permitted virus recovery from larger volumes of cells per experiment. Zonal centrifugation of concentrated cell‐free spent media from highly infected cell cultures yielded more purified virus than obtained from cells. Viral concentrates were prepared with particle counts of 10 10 –10 11 /ml and total protein concentrations of 0.2–0.5 mg/ml. Subsequent isopyenie‐zonal centrifugation of the various high‐count virus fractions from the zonal centrifuge showed a heterogeneity in buoyant virus density ranging from 1.18 to 1.27 in potassium tart rate. The spread in virus density was attributed to the different morphological forms of the virus observed by electron microscopy.