Expression, Purification And Activity Of Ferredoxin Nadp~+ Reductase From Acidithiobacillus Ferrooxidans

Ferredoxin(flavodoxin)-NADP(H) reductase(FNR) is a ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors(ferredoxin,flavodoxin,asrenodoxin) to redox based metabolisms in plastids,mitochondria and bacteria.The gene of ferredoxin NADP+ reductase(FNR) cloned from A. ferrooxidans ATCC23270 was ligated with PLM I which contains a T7 promoter by T4 ligase.T7 promoter is a promoter comes from T7 bacteriophage which has high specificity.Only T7 RNA ploymerase can makes it works so that cloned gene could be independent expressed, we use DH5 a (E.coli)as host to get high concentration and high purity recombined plasmid.Then use BL21 (E.coli) as host which cotains lacUV5 promoter that coding T7 RNA ploymerase to express high quality FNR, and the lack of Ion gene which coding a protease that can enzymolysis foreign protein is to the benefit of foreign DNA expression.we express FNR with his tag under advantage condition,and pure it with affinity chromatography.To pured FNR,the concentration was 1.441ug/ul measured by Coomassie Brilliant Blue,the molecular mass was 36KDa measured by SDS-PAGE.UV-vis scanning showed absorption maxima at 380 nm and 450 nm, suggesting the existence of FAD.And KmNADPH=0.01787mmol/L, VmaxNASPH=0.0329mmol/L/min KmEDTA-Fe5+=0.1437mmol/L, VmaxEDTA-Fe5+=0.0011mmol/L/min by Michaelis-Menten equation and double reciprocal plot.3D structure of FNR was simulated with Swiss-model. All above lay the foundation of advantage research of FNR from A. ferrooxidans such as characteristic and structure.