Steinemann D, Engelbrecht S, Lill H
Abt. Biophysik, Universität Osnabrück, Germany.
FEBS Lett. 1995 Apr 3;362(2):171-4. doi: 10.1016/0014-5793(95)00238-5.
Subunits alpha, beta, and gamma of the F1-part of cyanobacterial F0F1-ATPase have been cloned into expression vectors. Over-expressed subunit beta was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts. Recombinant alpha and gamma subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded. The correct folding and functional integrity of the alpha and beta subunits was monitored by their ability to bind nucleotides. Active cyanobacterial F1-ATPase was assembled from its purified subunits alpha, beta, gamma, delta and epsilon. The reassembled enzyme reconstituted ATP synthesis in F1-depleted thylakoid membranes of Synechocystis sp. PCC 6803 and hydrolyzed ATP.
蓝藻F0F1 - ATP酶F1部分的α、β和γ亚基已被克隆到表达载体中。在适当的培养和诱导条件下，发现过表达的β亚基可溶于大肠杆菌细胞的细胞质部分，并从细胞提取物中纯化出来。重组α和γ亚基沉淀形成包涵体，必须进行溶解、纯化和重折叠。通过α和β亚基结合核苷酸的能力监测其正确折叠和功能完整性。活性蓝藻F1 - ATP酶由其纯化的α、β、γ、δ和ε亚基组装而成。重新组装的酶在集胞藻PCC 6803的F1缺失类囊体膜中重建了ATP合成并水解了ATP。
