title: High-level expression of Mycoplasma arginine deiminase in Escherichia coli and its efficient renaturation as an anti-tumor enzyme.
abstract: 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.
authors.affiliation: Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, Saitama.
authors.forename: S
authors.initials: S
authors.lastname: Misawa
authors.forename: M
authors.initials: M
authors.lastname: Aoshima
authors.forename: H
authors.initials: H
authors.lastname: Takaku
authors.forename: M
authors.initials: M
authors.lastname: Matsumoto
authors.forename: H
authors.initials: H
authors.lastname: Hayashi
mesh_terms: 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
chemical_list: D000970:Antineoplastic Agents; D004269:DNA, Bacterial; D006867:Hydrolases; C019723:arginine deiminase