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

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2013 06
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Expression, Purification and Activity Determination of the Ferredoxin-NADP+ Reductase in Aeromonas hydrophila XS91-4-1
ZHANG Kai CHEN Huxing YU Jinhui YAN Jiali LIU Deli XIONG Li
(College of Life Sciences, Central China Normal University, Wuhan 430079, China)
Aeromonas hydrophila ferredoxin-NADP+ reductase (FNR) soluble expression protein purification enzyme activity 3D structure
Q554 : Q936

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 purified by nickel column affinity 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 and 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 specific activity for NADPH and EDTA-Fe3+ was 1.78 U/mg and 1.13 U/mg respectively, 29 and 22-fold higher after purification. 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.


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