Blinder D, Magasanik B
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
J Bacteriol. 1995 Jul;177(14):4190-3. doi: 10.1128/jb.177.14.4190-4193.1995.
We describe the purification of the product of the GLN3 gene of Saccharomyces cerevisiae and the demonstration that the purified product, Gln3p, binds specifically to the DNA sequences GATAAG and GATTAG, previously identified as nitrogen-responsive upstream activation sequences (UASN). When Gln3p is overproduced, it is released from the cells in a highly aggregated form incapable of specific binding to UASN. We used Gln3p tagged with six histidine codons at the 5' terminus and equipped with a galactose-inducible promoter to overproduce histidine-tagged Gln3p. The material was denatured, adsorbed to an Ni-nitrilotriacetic acid (NTA)-agarose column, eluted with imidazole, and after renaturation further purified on a gel filtration column. We then demonstrated the specific binding of the more than 90% pure Gln3p to the UASN by gel shift and footprinting methods.
我们描述了酿酒酵母GLN3基因产物的纯化过程，并证明纯化产物Gln3p能特异性结合DNA序列GATAAG和GATTAG，这两个序列先前被鉴定为氮响应上游激活序列（UASN）。当Gln3p过量产生时，它以高度聚集的形式从细胞中释放出来，无法与UASN特异性结合。我们使用在5'末端带有六个组氨酸密码子并配备半乳糖诱导型启动子的Gln3p来过量表达组氨酸标签的Gln3p。该物质经过变性，吸附到镍-次氮基三乙酸（NTA）琼脂糖柱上，用咪唑洗脱，复性后再通过凝胶过滤柱进一步纯化。然后，我们通过凝胶迁移和足迹法证明了纯度超过90%的Gln3p与UASN的特异性结合。
