Subunit structure of recombinant rat liver L-tryptophan 2,3-dioxygenase

Rat liver L-tryptophan 2,3-dioxygenase (TDO) with a histidine tag at the N-terminus was expressed in Escherichia coli JM109 harboring plasmid pUC18 carrying the full-length cDNA of TDO. The recombinant enzyme was purified to near homogeneity by employing conventional purification methods including nickel-chelate immobilized resin column chromatography. The purified enzyme had a turnover number per heme 303 min(-1) with similar spectral properties to those of native rat liver enzyme. SDS-PAGE of purified TDO preparation showed two distinct bands with molecular masses of 49 and 46 kDa. N-terminal sequence analysis of the components revealed that the 46-kDa species is shorter than the 49-kDa one by 19 amino acid residues including six histidine residues at the end. Thus, a limited proteolysis appeared to occur between Tyr(13) and Thr(14) of the original polypeptide chain. The construct of recombinant TDO with deletion of the N-terminal 13 residues gave a single band on SDS-PAGE with a molecular mass of about 46 kDa. The N-terminal truncation had no effect on the catalytic activity nor on the spectral properties. (C) 2002 Elsevier Science B.V. All rights reserved.