通过应用于磷酸甘油酸变位酶的t-降解子技术在酵母中设置蛋白质的分级水平。
Setting of graded levels of a protein in yeast by a t-degron technique as applied to phosphoglycerate mutase.
作者信息
Heidrich Katja, Fraenkel Dan G
机构信息
Dept, of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave, Boston MA 02115, USA.
出版信息
BMC Genet. 2002 Jul 30;3:13. doi: 10.1186/1471-2156-3-13.
BACKGROUND
Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead.
RESULTS
In a yeast t-degron (ubiquitin-argDHFR(ts))- phosphoglycerate mutase (GPM1) fusion strain, increasing periods of exposure to the non-permissive temperature 37 degrees C, even in the presence of cycloheximide, gave decreasing function, as assessed at 23 degrees C in vivo by glucose metabolism and confirmed by immunoblot.
CONCLUSION
An ideal system would set a range of lower levels of a protein, do so without compensating protein synthesis, and give stable activity for in vitro comparisons. Although the first two aims appear obtainable, the third was not in this example of the application, limiting its uses for some but not all purposes.
背景
通过可控基因表达为体内研究设定蛋白质的分级水平存在不便之处，因此我们在此探索使用t-降解子技术取而代之。
结果
在酵母t-降解子（泛素-精氨酸二氢叶酸还原酶（ts））-磷酸甘油酸变位酶（GPM1）融合菌株中，即使存在环己酰亚胺，延长暴露于非允许温度37℃的时间，功能也会下降，在23℃下通过体内葡萄糖代谢评估，并通过免疫印迹法得到证实。
结论
一个理想的系统应该能够设定一系列较低水平的蛋白质，在不补偿蛋白质合成的情况下做到这一点，并为体外比较提供稳定的活性。尽管前两个目标似乎可以实现，但在这个应用实例中第三个目标未能实现，这限制了它在某些但不是所有目的上的用途。
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