CIRCULAR-DICHROISM AND ULTRAVIOLET-ABSORPTION OF A DEOXYRIBONUCLEIC-ACID BINDING-PROTEIN OF FILAMENTOUS BACTERIOPHAGE
CIRCULAR-DICHROISM AND ULTRAVIOLET-ABSORPTION OF A DEOXYRIBONUCLEIC-ACID BINDING-PROTEIN OF FILAMENTOUS BACTERIOPHAGE
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DOI：
10.1021/bi00750a017
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发表时间：
1973-01-01
期刊：
影响因子：
2.9
通讯作者：
DAY, LA
中科院分区：
文献类型：
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作者：
DAY, LA
材料和方法采用G5蛋白的几种制备方法。制剂G5A、G5B和G5D由Bruce M.Alberts和A.Keith Dunker作为冷冻溶液获得，制备G5C从Rolf Knippers获得冻干。用Alberts等(1972)的方法从感染了野生型Fd噬菌体的大肠杆菌K12 3300中分离出所有其他制剂，并对A.Keith Dunker提出的方法进行了修改。感染细胞在-20℃下冷冻保存，解冻，用缓冲液I(10 mMŧ，2 mM CaClO，1 mM/3-巯基乙醇，1 mM EDTA在0.02MTris-HCl中，pH 8.1)洗涤，并用玻璃珠在高速摇床中破碎。提取液，大约20ml/g湿细胞，在含有20ĩg/ml的胰腺核糖核酸酶(沃辛顿生化，批号693)和20克/毫升的胰腺脱氧核糖核酸酶1(沃辛顿生化，批号D1227-30)的情况下，在4℃孵育3小时。然后在氯化钠中煮1m，再孵化1小时，然后在10000克的离心机中离心20分钟。在室温下，每5.5毫升的乙醇提取液中加入4.5毫升饱和的(NH^SCL)。先沉淀物在冰浴中形成1小时，在10,000克的温度下旋转20分钟，然后再悬浮在每克湿细胞中约1毫升的冷缓冲液II(1毫升-巯基乙醇，5毫米EDTA，0.05ĩ氯化钠，以及10%V/V甘油在0.02 MTris-HCl中，pH 8.1)中，并在4℃对缓冲液II进行过夜透析。在透析后，样品在30,000g澄清3小时，并应用于DNA-纤维素柱(Alberts and Herrick，1970)与缓冲液II平衡的鲑鱼精子DNA(A级，加州生物化学，批号010021)和Whatman CF11纤维素制备脱氧核糖核酸。一根2.5×10 cm的单柱保持在4，用于几种G5蛋白制剂。在样品上样和用缓冲液II洗涤后，用含有0.4、0.8和2M氯化钠的缓冲液II分步洗脱。在十二烷基硫酸钠和十二烷基硫酸钠存在下，用0.8M氯化钠缓冲液II洗脱的物质经聚丙烯酰胺凝胶电泳法测定，除少数前导和拖尾部分外，其余均一。
Materials and MethodsSeveral preparations of G5 protein were used. Prepara-tions G5A, G5B, and G5D were received from Bruce M. Alberts and A. Keith Dunker as frozen solutions, and prep-aration G5C was received lyophilized from Rolf Knippers. All other preparations were isolated from Escherichia coli K12 3300 infected with wild-type fd bacteriophage by a method based on that of Alberts et al.(1972), with some modifications suggested by A. Keith Dunker. Infected cells were stored frozen at—20, thawed, washed with buffer I (10 mM MgClŧ, 2 mM CaClo, 1 mM/3-mercaptoethanol, and 1 mM EDTA in 0.02 m Tris-HCl, pH 8.1), and disrupted with glass beads in a high-speed shaker. The extract, approximately 20 ml/g of wet cells, was incubated for 3 hr at 4 in the presence of 20 ĩg/ml of pancreatic ribonuclease (Worthington Biochemical, lot 693) and 20 gg/ml of pancreatic deoxyribonuclease 1 (Worthington Biochemical, lot D1227-30). It was then made 1 m in NaCl, incubated for 1 more hr, and centrifuged at 10,000 g for 20 min. To each 5.5 ml of coldextract solution was added 4.5 ml of saturated (NH^ SCL at room temperature. Pre-cipitate was allowed to develop for 1 hr in an ice bath, spun down at 10,000 gfor 20 min, and resuspended in approximately1 ml of cold buffer II (1 mM ß-mercaptoethanol, 5 mM EDTA, 0.05 ĩ NaCl, and 10% v/v glycerol in 0.02 m Tris-HCl, pH 8.1) per g of wet cells and dialyzed overnight at 4 against buffer II. After dialysis, the sample was clarified at30, 000g for 3 hr and applied to a DNA-cellulose column (Alberts and Herrick, 1970) equilibrated with buffer II. Salmon sperm DNA (A grade, Calbiochem, lot 010021) and Whatman CF11 cellulose were used toprepare the DNA-cellulose. A single column measuring 2.5 X 10 cm maintained at 4 was used for several G5 protein preparations. After sample application and washing with buffer II, the column was eluted by steps with buffer II containing 0.4, 0.8, and 2 m NaCl. Except for a few leading and trailing fractions, the material eluted with 0.8 m NaCl-buffer II was homogeneous by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfateand