Growth inhibition of human melanoma cells by a recombinant arginine deiminase expressed in Escherichia coli.
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.
Kozai
Megumi
M
Department of Veterinary Microbiology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan.
Sasamori
Eriko
E
Fujihara
Masatoshi
M
Yamashita
Tetsuro
T
Taira
Hideharu
H
Harasawa
Ryô
R
D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D008545:Melanoma / Q000378:metabolism*; D019535:Mycoplasma hominis / Q000201:enzymology*; D011994:Recombinant Proteins
D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D006867:Hydrolases; C019723:arginine deiminase