DOI: 10.3724/SP.J.1145.2013.01003

Chinese Journal of Appplied Environmental Biology (应用与环境生物学报) 2013/19:6 PP.1003-1007

Expression, Purification and Activity Determination of the Ferredoxin-NADP+ Reductase in Aeromonas hydrophila XS91-4-1

Ferredoxin-NADP+ reductases(FNRs) are ubiquitous flavoenzymes that play an important role in many organisms. To investigate the structure and function of Aeromonas hydrophila FNR, FNR gene was cloned from A. hydrophila XS91-4-1. Recombinant plastimid pET42a-fnr was constructed and overexpressed in Escherichia coli BL21. FNR-GST recombinant protein was purif ied by nickel column affi nity chromatography. According to Michaelis-Menten equation and double reciprocal plot, the enzyme activity of recombinant protein was assayed using NADPH and EDTA-Fe3+ as substrate. Then bioinformatics analysis of FNR was performed a nd three-dimensional structure of FNR was predicted. The results showed that FNR-GST recombinant protein was highly expressed in E. coli BL21 in a soluble form. Its protein concentration was 67.3 μg/mL. The specif ic activity for NADPH and EDTAFe3+ was 1.78 U/mg and 1.13 U/mg respectively, 29 and 22-fold higher after purif ication. Based on its sequence and phylogenetic relationship, the FNR of A. hydrophila XS91-4-1 was closely related to bacterial-class FNR. Our study suggested that the FNR of A. hydrophila belongs to bacterial-class FNR, and is similar to FNRs in many fundamental characteristics. Fig 6, Tab 1, Ref 29

Key words:Aeromonas hydrophila,ferredoxin-NADP+ reductase (FNR),soluble expression,protein purification,enzyme activity,3D structure

ReleaseDate:2015-04-15 08:42:22

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