Davasse V, Moulis J M
Département de Biologie Moléculaire et Structurale, Laboratorie Métalloprotéines, C.E.N.G., Grenoble, France.
Biochem Biophys Res Commun. 1992 May 29;185(1):341-9. doi: 10.1016/s0006-291x(05)80991-x.
A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.
已合成了一个编码巴氏梭菌2[4Fe-4S]铁氧化还原蛋白精确序列且含有11个独特限制性内切酶切割位点的基因，并将其克隆到大肠杆菌中。该合成基因在大肠杆菌中高效表达，其产物已被纯化和表征。N端序列与从巴氏梭菌中分离的蛋白质相同，重组铁氧化还原蛋白含有与均一全铁氧化还原蛋白预期相同数量的[4Fe-4S]簇（每个单体2个）。作为巴氏梭菌氢化酶I的电子供体，它显示出与天然铁氧化还原蛋白相同的还原电位和动力学参数。所有这些特性表明，在大肠杆菌中表达的2[4Fe-4S]铁氧化还原蛋白与亲本梭菌蛋白相同。
