Dreiseikelmann B, Velleman M, Schuster H
Max-Planck-Institut für Molekulare Genetik, Berlin, Federal Republic of Germany.
J Biol Chem. 1988 Jan 25;263(3):1391-7.
The c1 repressor gene of bacteriophage P1 is located on P1 DNA EcoRI fragment 7 (Sternberg, N. (1979) Virology 96, 129-142). Subfragments of P1 DNA EcoRI fragment 7 were cloned into expression vectors, and the c1 repressor protein from P1 wild-type phage and a revertant of a temperature-sensitive repressor mutant were overproduced in Escherichia coli and purified to near-homogeneity. The decreased electrophoretic mobility of P1 DNA BamHI fragment 9 in the presence of appropriate protein fractions was used as an assay for the repressor protein. Highly purified repressor migrates as a single polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide gels, corresponding to a molecular weight of about 33,000. A molecular weight of about 63,000 for the native repressor molecule was calculated from determinations of the sedimentation coefficient, which was 2.6 s, and the Stokes radius, which was 55 A. Cross-linking the protein with glutaraldehyde yielded two bands. These data and a high frictional coefficient (2.1) suggest that the native repressor exists in solution as an asymmetric dimer molecule.
噬菌体P1的c1阻遏基因位于P1 DNA的EcoRI片段7上（斯特恩伯格，N.（1979年）《病毒学》96，129 - 142）。将P1 DNA的EcoRI片段7的亚片段克隆到表达载体中，P1野生型噬菌体和温度敏感型阻遏突变体的回复体中的c1阻遏蛋白在大肠杆菌中过量表达，并纯化至接近均一。在存在适当蛋白质组分的情况下，P1 DNA的BamHI片段9电泳迁移率降低被用作阻遏蛋白的检测方法。高度纯化的阻遏蛋白在变性十二烷基硫酸钠 - 聚丙烯酰胺凝胶上作为单一多肽迁移，对应分子量约为33,000。根据沉降系数（2.6 s）和斯托克斯半径（55 Å）的测定，计算出天然阻遏分子的分子量约为63,000。用戊二醛使蛋白质交联产生两条带。这些数据和高摩擦系数（2.1）表明天然阻遏蛋白在溶液中以不对称二聚体分子形式存在。
