van Criekinge W, Cornelis S, Van De Craen M, Vandenabeele P, Fiers W, Beyaert R
Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Belgium.
Mol Cell Biol Res Commun. 1999 May;1(2):158-61. doi: 10.1006/mcbr.1999.0125.
Yeast two-hybrid technology as well as mammalian reporter assays use fusions between a protein of interest and the GAL4 DNA-binding domain (GAL4DB). We demonstrate that expression of a GAL4DB/caspase-1 chimeric protein in yeast leads to autoproteolytic cleavage of GAL4DB. Moreover, recombinant GAL4DB is a good in vitro substrate for recombinant caspase-1 and several other caspases. Cleavage sites map at the C-terminus of GAL4DB and result in release of the fused protein. The finding that GAL4DB can be cleaved by caspases has important implications for the use of caspases in two-hybrid analysis and in the interpretation of mammalian assays based on GAL4-dependent reporter gene expression.
酵母双杂交技术以及哺乳动物报告基因检测使用感兴趣的蛋白质与GAL4 DNA结合结构域（GAL4DB）之间的融合体。我们证明，在酵母中表达GAL4DB/半胱天冬酶-1嵌合蛋白会导致GAL4DB的自蛋白水解切割。此外，重组GAL4DB是重组半胱天冬酶-1和其他几种半胱天冬酶的良好体外底物。切割位点位于GAL4DB的C末端，导致融合蛋白的释放。GAL4DB可被半胱天冬酶切割这一发现对于在双杂交分析中使用半胱天冬酶以及基于GAL4依赖性报告基因表达的哺乳动物检测的解释具有重要意义。
