J Integr Plant Biol. ›› 1998, Vol. 40 ›› Issue (9): -.

• Research Articles •    

Overexpression of petH Gene of Cyanobacterium synechococcus sp. PCC 7002 in Escherichia coll and Purification of the Expressed Product

LI Rong-Gui, ZHAO Jin-Dong, WU Guang-Yao and WU Xiang-Yu   

Abstract: Fd:NADP+ oxidoreductase (FNR) is one of the key enzymes in photosynthetic electron transport. The gene petH encoding FNR of Synechococcus sp. PCC 7002 was cloned into the expressing vector pET-3 d' and overexpressed in E. coli. The amount of recombinant FNR (rFNR) was over 50% of the total cellular proteins. There were two forms of FNR activity, one is soluble and the other one was in the form of inclusion bodies. The soluble rFNR was purified through ion exchange chromatography and gel chromatography. The rFNR in the form of inclusion bodies was first solubilized with 6.7 mol/L urea, and then refolded into the active form in the presence of flavin adenine dinucleotide (FAD). Further purification was performed by ion exchange chromatography. The rFNR pmified from either form of the expressed product had the maximum absorption spectrum as that of the natural FNR from cyanobacteria, whose maximum absorption was at 273, 385 and 456 ran respectively. N-tenninal sequencing showed that rFNR was indeed a product of petH gene expression, rFNR could catalyze the electron transport from P700 to NADP+ in the presence of ferredoxin. The optimal pH for diaphorase activity of rFNR was 8.0 and the optimal temperature was 30 ℃.

Key words: petH gene, FNR, Electron transport, Overexpression, Protein refolding

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