Norais N, Vaillier J, Velours J
Institut de Biochimie Cellulaire du CNRS, Université de Bordeaux II, France.
Biochem Biophys Res Commun. 1994 Apr 29;200(2):877-83. doi: 10.1006/bbrc.1994.1532.
The yeast ATP synthase subunit d was over-expressed in E. coli and formed inclusion bodies. It was purified by solubilization in urea and slow removal of the urea by stepwise dialysis in the presence of a non-ionic detergent. The resulting soluble subunit d was used to prepare polyclonal antibodies. Blots of yeast mitochondrial proteins were probed with these antibodies. The strain disrupted in ATP4 gene encoding the subunit 4 displayed only 8% of the wild type subunit d. Antibodies against subunit d did not inhibit the wild type ATPase activity.
酵母ATP合酶亚基d在大肠杆菌中过表达并形成包涵体。通过在尿素中溶解并在非离子去污剂存在下逐步透析缓慢去除尿素来进行纯化。所得的可溶性亚基d用于制备多克隆抗体。用这些抗体探测酵母线粒体蛋白的印迹。编码亚基4的ATP4基因被破坏的菌株仅显示野生型亚基d的8%。针对亚基d的抗体不抑制野生型ATP酶活性。
