Fennewald M A, Gerrard S P, Chou J, Casadaban M J, Cozzarelli N R
J Biol Chem. 1981 May 25;256(10):4687-90.
The transposase encoded by the tnpA gene of Tn3 is a protein specifically required for Tn3 transposition. We have purified it to homogeneity from an Escherichia coli strain containing a mutant Tn3 that overproduces transposase. About a 10-fold additional increase in transposase resulted from growth into stationary phase. The initial purification was guided by the presence of a protein band with the electrophoretic mobility of the tnpA gene product. The identity of the purified protein was proven by the agreement of five NH2-terminal amino acids with the nucleotide sequence of the A gene; this, in turn, fixed the initiation codon. Transposase formed large aggregates in the absence of Mg2+ at salt concentrations of 0.1 M or less. In nonaggregating conditions, it had 1 or 2 copies of 113,000-dalton protomers. Subsequent purifications exploited the rapid and simple assay of transposase-mediated retention of labeled DNA to a nitrocellulose filter. Transposase bound tightly to single-stranded DNA but weakly to intact duplex DNA. DNA binding did not require Mg2+ and was highly salt-resistant. Binding did not require specific sequences, because poly(dT) was as good a substrate as phi X174 viral DNA. The high DNA binding constant of 4 X 10(9) M-1 is about the same as for some single-stranded DNA binding proteins.
Tn3的tnpA基因编码的转座酶是Tn3转座所特需的一种蛋白质。我们从含有过量产生转座酶的突变型Tn3的大肠杆菌菌株中，将其纯化至同质状态。进入稳定期后，转座酶产量增加了约10倍。最初的纯化是根据具有tnpA基因产物电泳迁移率的蛋白条带进行的。纯化蛋白的身份通过其5个氨基末端氨基酸与A基因核苷酸序列的一致性得到证实；这反过来又确定了起始密码子。在盐浓度为0.1M或更低且不存在Mg2+的情况下，转座酶形成大聚集体。在非聚集条件下，它有1个或2个113,000道尔顿的原体拷贝。后续的纯化利用了转座酶介导的标记DNA保留在硝酸纤维素滤膜上的快速简便检测方法。转座酶与单链DNA紧密结合，但与完整的双链DNA结合较弱。DNA结合不需要Mg2+，并且具有高度的耐盐性。结合不需要特定序列，因为聚（dT）与phi X174病毒DNA一样是良好的底物。4×10^9 M-1的高DNA结合常数与一些单链DNA结合蛋白的结合常数大致相同。
