Reha-Krantz L J, Hurwitz J
J Biol Chem. 1978 Jun 10;253(11):4043-50.
The dnaB gene product was purified to homogeneity and its physical properties were characterized. Purification was aided by the use of the Escherichia coli strain. MV12/28, which overproduced the dnaB gene product 10-fold (Wickner, S. H., Wickner, R. B., and Raetz, C. R. H. (1976) Biochem. Biophys. Res. Commun. 70, 389-396) and by taking advantage of the enzyme's high affinity for both DEAE-cellulose and phosphocellulose. The most highly purified fractions gave a single stained band on native, polyacrylamide gels and dnaB enzymatic activity was coincident with this band. On denaturing sodium dodecyl sulfate-polyacrylamide gels, a single band was observed corresponding to a molecular weight of 48,000 +/- 2,000. The native molecular weight of 290,000 +/- 12,000 was calculated from determinations of the sedimentation coefficient, which was 11.3 S, and the Stokes radius, which was 60 A. Cross-linking the protein with dimethyl suberimidate yielded six bands. We conclude that the enzyme consists of six identical subunits. The apparent pI was 4.9 and the amino acid composition was typical except for the absence of cysteine.
将dnaB基因产物纯化至同质，并对其物理性质进行了表征。使用大肠杆菌菌株MV12/28辅助纯化，该菌株使dnaB基因产物过量表达10倍（维克纳，S.H.，维克纳，R.B.，和雷茨，C.R.H.（1976年）《生物化学与生物物理研究通讯》70，389 - 396），并利用该酶对DEAE - 纤维素和磷酸纤维素的高亲和力。纯化程度最高的级分在天然聚丙烯酰胺凝胶上呈现一条染色带，且dnaB酶活性与这条带重合。在变性十二烷基硫酸钠 - 聚丙烯酰胺凝胶上，观察到一条对应分子量为48,000±2,000的带。根据沉降系数（为11.3 S）和斯托克斯半径（为60 Å）的测定，计算出天然分子量为290,000±12,000。用辛二酸二甲酯亚胺使蛋白质交联产生六条带。我们得出结论，该酶由六个相同的亚基组成。表观pI为4.9，氨基酸组成除不含半胱氨酸外是典型的。
