Abstract
Nitrate reductase (NR) is a central enzyme in nitrogen metabolism in plants. However, NR protein is small amounts in plants, which makes recombinant expression an attractive approach for studying its biochemistry.
NR CcR+ fragemnt (43 kDa fragment containing FAD, Fe-heme and Hinge-1 including regulatory Ser residue) was expressed in yeast (Pichia pastoris). CcR+ fragment was expressed only 1 % of CcR- (41 kDa fragment without the Ser residue ) fragment. We modified nucleotide sequence in the front of ATG codon in the cDNA fragment coding CcR+. This modified nucleotide sequence was same as that of the cDNA fragment coding CcR-. This CcR+ fragment was expressed about 59 mg/L Pichia culture. Next, we modified full length NRcDNA in the same manner. Active NR protein was expressed about 0.7 mg/L culture and this amount of NR protein was ten times higher than that of unmodified one.
