High-level expression of Mycoplasma arginine deiminase in Escherichia coli and its efficient renaturation as an anti-tumor enzyme.
The arginine deiminase (AD) gene was cloned from Mycoplasma arginini and expressed in the cytosol of Escherichia coli as inclusion bodies with an expression level of at least 20% of the total bacterial proteins. The inclusion bodies were solubilized with 6 M guanidine hydrochloride (Gdn-HCl) under reducing conditions, in order to avoid incorrect disulfide-bond formation of the recombinant (r-) AD molecules, and renaturation was performed under various refolding conditions. The optimum renaturation conditions were found to be incubation for 90 h at pH 7.5 and 15 degrees C. The resulting completely refolded r-AD was purified to homogeneity by anion-exchange and arginine-affinity chromatography and its activity yield was 72.5%. The specific activity of the purified r-AD was comparable to and its amino acid composition was identical to those of Mycoplasma AD, and NH2-terminal sequence analysis revealed that its methionine residue corresponding to the translation initiation codon had been removed completely. Anti-tumor activity analyses showed that r-AD inhibited the growth of two mouse cell lines, hepatoma MH134 and fibrosarcoma Meth A, strongly in vitro at concentrations in excess of 10 ng ml-1. Moreover, when MH134-implanted mice were given single intravenous injections of r-AD at doses of 50 mg kg-1 and higher, their survival times were prolonged significantly. These results, taken together, indicate that the enzymatic properties and biological actions of r-AD were highly consistent with those of Mycoplasma AD.
Misawa
S
S
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, Saitama.
Aoshima
M
M
Takaku
H
H
Matsumoto
M
M
Hayashi
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006867:Hydrolases / Q000235:genetics* / Q000494:pharmacology; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000201:enzymology* / Q000235:genetics*; D009374:Neoplasms, Experimental / Q000188:drug therapy; D011487:Protein Conformation
D000970:Antineoplastic Agents; D004269:DNA, Bacterial; D006867:Hydrolases; C019723:arginine deiminase