Homma T, Yoshihisa T, Kihara A, Akiyama Y, Ito K
Department of Cell Biology, Kyoto University, Japan.
Biochem Biophys Res Commun. 1995 Oct 13;215(2):452-8. doi: 10.1006/bbrc.1995.2486.
Intracellular stability of alpha fragments of beta-galactosidase in Escherichia coli has been studied by pulse-chase/immunoprecipitation experiments. An alpha fragment encoded by the pUC118 vector was relatively stable with an estimated half-life of about 12 min at 37 degrees C, whereas another vector, pSTV28, encoded a less stable alpha fragment that had a different carboxy-terminal sequence. Stability of the fragment was found to be affected markedly by amino-terminal attachment of other sequences. An amino-terminal fusion of a sequence derived from cytoplasmic domain 4 of the SecY protein shortened the half-life of the alpha fragment to less than 1 min. In contrast, an amino-terminal sequence from the NusG protein had no apparent effect on the stability of the fragment. In a fusion protein in which the intact SecY protein was fused to the alpha fragment, stabilization of the SecY part by overproduction of the partner SecE protein resulted in an increased alpha complementation activity of beta-galactosidase. These results indicate that stability of alpha fragment can be dictated by the stability of the fused protein. The alpha fragment of beta-galactosidase, which is unique in that it is largely unstructured but can be "active" in alpha complementation, may be used as an in vivo indicator of stability of proteins attached to it.
通过脉冲追踪/免疫沉淀实验研究了大肠杆菌中β-半乳糖苷酶α片段的细胞内稳定性。由pUC118载体编码的α片段相对稳定，在37℃时估计半衰期约为12分钟，而另一个载体pSTV28编码的α片段稳定性较差，其羧基末端序列不同。发现该片段的稳定性受到其他序列氨基末端连接的显著影响。源自SecY蛋白细胞质结构域4的序列的氨基末端融合将α片段的半衰期缩短至不到1分钟。相反，来自NusG蛋白的氨基末端序列对该片段的稳定性没有明显影响。在完整的SecY蛋白与α片段融合的融合蛋白中，通过过量表达伴侣SecE蛋白使SecY部分稳定，导致β-半乳糖苷酶的α互补活性增加。这些结果表明，α片段的稳定性可由融合蛋白的稳定性决定。β-半乳糖苷酶的α片段独特之处在于它基本上是无结构的，但在α互补中可以“有活性”，它可以用作与其相连蛋白质稳定性的体内指示剂。
