Purification and characterization of the SV40 F-gene protein.
Purification and characterization of the SV40 F-gene protein.
复制标题
SV40 F 基因蛋白的纯化和表征。
DOI：
10.1101/sqb.1980.044.01.018
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
Schuetz,FR
中科院分区：
文献类型：
--
作者：
Tegtmeyer,P;Spillman,T;Schuetz,FR
方法制备细胞提取物。在100/~ Ci/ml [35S]蛋氨酸的持续存在下，SV40的SVS菌株以10倍的倍数感染CV-1细胞。37~ 72小时后，用等渗缓冲液冲洗细胞，在0.01 M MES （Calbiochem）、0.01 M NaCI、0.004 M MgCI2 （pH 6）中肿胀。用Dounce匀浆法破坏细胞，在2000g时将细胞分离成细胞质和细胞核部分。细胞核在0.01 M HEPPS (Calbiochem), 0.01 M NaCI （pH 8）中重悬。将Dounce细胞质和重悬细胞核在10万g下旋转30分钟，4~用1% Triton X-100在pH为8的等渗缓冲液中重新提取。硫酸铵沉淀。将pH为8的饱和（NI-I4） 2-SO4加入到Dounce细胞质的上清部分中，最终浓度为50%。提取液以20,000 g纺丝1小时。在0.01 M Tris, 0.001 M EDTA （pH 8）中，用20%(NI-I4) 2SO4重悬球，如上所述进行纺丝。上清部分含有f基因蛋白。
METHODSPreparation of cellular extracts. CV-1 cells were infected with the SVS strain of SV40 at a multiplicity of 10 in the continuous presence of 100/~ Ci/ml of [35S] methionine. After 72 hours at 37~ the cells were washed with isotonic buffer and swollen in 0.01 M MES (Calbiochem), 0.01 M NaCI, 0.004 M MgCI2 (pH 6). The cells were disrupted by Dounce homogenization and separated into cytoplasmic and nuclear fractions at 2000g. The nuclei were resuspended in 0.01 M HEPPS (Calbiochem), 0.01 M NaCI (pH 8). The Dounce cytoplasm and the resuspended nuclei were spun at 100,000 g for 30 minutes at 4~ The pellets were rcextracted with 1% Triton X-100 in isotonic buffer at pH 8.Ammonium sulfate precipitation. Saturated (NI-I4) 2-SO4 at pH 8 was added to the supernatant fraction of the Dounce cytoplasm to a final concentration of 50%. The extracts were spun for 1 hour at 20,000 g. The pellet was resuspended in 20%(NI-I4) 2SO4 in 0.01 M Tris, 0.001 M EDTA (pH 8) and spun as above. The supernatant fraction contained the F-gene protein.