ABSTRACT. We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361. Arginine dehydrolysing mycoplasmas convert L-arginine into L-citrulline and ammonia by the arginine deiminase (ADI; EC 3.5.3.6) activity [1]. ADI gene of Mycoplasma hominis PG21 has been cloned in Escherichia coli by using plasmid pUC118 [7]. ADI has been shown to affect apoptosis and inhibit NO synthesis (i.e., anti-angiogenic effects), and exert effects against tumor necrosis factor- (TNF-) and neutralize endotoxin. [2,6,11,16,23,27]. ADI has also been known to inhibit the growth of melanoma and hepatocellular carcinoma cells in vitro as well as in vivo [19,20,24,26]. This anti-tumor activity of ADI has been attributed to the depletion of L-arginine, which is essential in these tumor cells [20,25]. Normal cells are not sensitive to external addition of ADI since they produce L-arginine by themselves. Expression of the ADI gene of M. homins PG21 in E. coli has been hampered since the ADI gene of M. hominis has four opal nonsense codons, which are known to encode tryptophan in mycoplasma cells. We have changed these opal codons to universal tryptophan codons and successfully expressed the recombinant ADI (rADI) gene of M. homins in E. coli, and demonstrated the enzymatic activity of purified rADI.
MATERIALS AND METHODS
Construction of ADI expression vector:Four stop codons TGA in the ADI gene cloned in pUC118 was changed into TGG by site-directed mutagenesis mediated by DpnImethod in order to express in E. coli [21]. Correct introductions of the mutation were confirmed by DNA sequencing. The mutated ADI gene, designated as SYN1903, was recloned into pET-47b (Novagen, Darmstadt, Germany) expression vector.Expression and purification of ADI: Recombinant pET47b plasmids encoding mycoplasmal ADI gene were transformed into competent E. coli BL21 (DE3) cells. The transformant was grown in 600 ml Luria broth (Sigma-Aldrich, St. Louis, MO, U.S.A.) at 30C, and isopropyl 1-thio--Dgalactoside was added at final concentration of 0.1 mM for induction of t...
