Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.
Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.
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DOI：
10.1073/pnas.80.22.6848
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发表时间：
1983-11
影响因子：
11.1
通讯作者：
J. Germino;J. Gray;H. Charbonneau;T. Vanaman;D. Bastia
中科院分区：
文献类型：
--
作者：
J. Germino;J. Gray;H. Charbonneau;T. Vanaman;D. Bastia
质粒 R6K 的 DNA 复制起始是由 35 千道尔顿的起始蛋白触发的。由于其不稳定且缺乏方便的测定程序来帮助其纯化，引发蛋白一直难以捉摸。使用重组DNA技术，我们将起始子的顺反子在其COOH末端附近以正确的阅读框融合到大肠杆菌的lacZ顺反子上从NH2末端算起的第九个密码子处。融合的顺反子产生的蛋白质不仅在体内稳定，而且具有双重活性：在体内和体外启动DNA复制以及β-半乳糖苷的水解。使用对 β-半乳糖苷酶具有特异性的亲和柱，我们已经证明可以快速纯化杂合蛋白，使其接近均质。利用引发剂的聚合结构，我们还分离了引发剂蛋白的非融合形式，其通过与β-半乳糖苷酶融合蛋白的亚基相互作用相关联，这使得可以通过亲和层析对其进行纯化。杂聚物的NH2末端氨基酸序列分析不仅表明融合和非融合的起始子具有相同的序列，而且还证实了从其核苷酸序列预测的起始子的蛋白质序列。这里描述的技术通常可用于分离难以通过常规程序纯化的其他蛋白质。
The initiation of DNA replication of plasmid R6K is triggered by a 35-kilodalton initiator protein. The initiator protein had been elusive because of its lability and the lack of a convenient assay procedure to aid its purification. Using recombinant DNA techniques, we have fused the cistron of the initiator near its COOH-terminal end, in the correct reading frame, to the lacZ cistron of Escherichia coli at the ninth codon from the NH2 terminus. The fused cistron yielded a protein that was not only stable in vivo but also had dual activities: initiation of DNA replication in vivo and in vitro and hydrolysis of beta-galactoside. Using an affinity column that is specific for beta-galactosidase, we have demonstrated the rapid purification of the hybrid protein to near homogeneity. Exploiting the polymeric structure of the initiator, we have also isolated the nonfused form of the initiator protein, associated through subunit interaction with the beta-galactosidase-fused protein, which permits its purification by affinity chromatography. NH2-terminal amino acid sequence analysis of the heteropolymer has not only shown that the fused and nonfused initiators have the same sequence but also confirmed the protein sequence of the initiator as predicted from its nucleotide sequence. The techniques described here should be generally useful for the isolation of other proteins that are difficult to purify by conventional procedures.