Biochem. J. (1990) 270, 697-703 (Printed in Great Britain)
697
Expression of human liver arginase in Escherichia coli Purification and properties of the product Masaki IKEMOTO,* Masayoshi TABATA,* Toshio MIYAKE,t Toshiaki KONO,t Masataka MORI,4 Masayuki TOTANI§II and Takashi MURACHI§T *College of Medical Technology, Kyoto University, Kyoto 606, tBiotechnology Research Laboratory, TOSOH Corp., Ayase, Kanagawa-ken 252, llnstitute for Medical Genetics, Kumamoto University Medical School, Kumamoto 862, and §Department of Clinical Science and Laboratory Medicine, Kyoto University Faculty of Medicine, Kyoto 606, Japan
Arginase is an enzyme that catalyses the hydrolysis of arginine to urea and ornithine. It is abundantly present in the liver of ureotelic animals (i.e. those whose excretion is characterized by the excretion of uric acid as the chief end-product of nitrogen metabolism), but its purification has hitherto not been simple, and the yield not high. Starting with a partially truncated cDNA for human liver arginase recently made available, we constructed an expression plasmid that had tandemly linked tac promotors placed upstream of a full-length cDNA. By selecting Escherichia coli strain KY1436 as the host micro-organism, we established an efficient system for the production of human liver arginase protein. Chromatographies on CM-Sephadex G-150, DEAE-cellulose and Sephadex G-150, followed by preparative agar-gel electrophoresis, yielded 10 mg of apparently homogeneous enzyme protein from 1 g (wet wt.) of E. coli cells. E. coliexpressed human liver arginase had chemical, immunological and most catalytic properties indistinguishable from those of purified human erythrocyte arginase. However, E. coli-expressed arginase was a monomer of Mr 35000, whereas the purified erythrocyte arginase was trimer of Mr 105 000. They differed also in pH- and temperature-stabilities. Gel-filtration experiments with these two purified arginases under various conditions, as well as with unfractionated human liver and erythrocyte cytosol preparations, indicated that the native form of human arginase should be of Mr 35000, and that the trimeric appearance of human erythrocyte arginase after purification was an artifact of the purification procedures. It was thus concluded that, in Nature, the liver and erythrocyte arginases are identical proteins.
INTRODUCTION Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the final step of urea formation in the mammalian liver. Despite its abundance in the liver, purification of human liver arginase has been reported only with rather poor yield and purity (Bascur et al., 1966; Beruter et al., 1978) or with complicated methods (Greenberg et al., 1956; Beruter et al., 1978). Human erythrocytes have been used as a better starting material for the purification of human arginase, yielding an apparently homogeneous preparation of arginase, though again by rather tedious procedures (Nishibe, 1973). Moreover, no conclusive evidence has been obtained for the identity of this purified erythrocyte arginase with liver arginase. In addition, a fairly large amount of human liver arginase as a pure protein is needed, not only for biochemical studies, but also for more practical purposes. For example, the release of liver arginase into plasma has been reported to be useful as a diagnostic index of liver damage, such as that caused by carbon tetrachloride (Kaiju, 1938; Utzino & Ogawa, 1957; Ogawa, 1958). For developing an appropriate immunoassay system for laboratory diagnosis, pure antigen arginase is a prerequisite. Also, several cases of hyperargininaemia have been reported to reflect a hereditary deficiency of liver arginase (Cederbaum & Shaw, 1977; Spector et al., 1983; Bernar et al., 1986); in such cases human liver arginase may find a potential therapeutic application. Molecular cloning and nucleotide sequence of cDNA for human liver arginase has been reported, but the longest cDNA used for sequencing still lacked 24 nucleotides at its 5' end, thus yielding, when expressed in Escherichia coli, an arginase-like
product that had only 0.0180% of the enzymic activity of the native enzyme (Haraguchi et al., 1987). We therefore attempted to construct an expression plasmid housing a full-length cDNA for human liver arginase, and established a system for producing arginase protein that yielded as much as 10 mg of purified arginase per 1 g wet weight E. coli cells. The product, after purification by relatively simple procedures, was characterized in comparison with the human erythrocyte arginase that we had purified earlier (Ikemoto et al., 1989). Such a comparison led us to conclude that human liver and erythrocyte arginases are identical proteins, both having Mr values of 35 000 in their native states.
MATERIALS AND METHODS Materials CM Sephadex C-50, Sephadex G-150, Sephadex G-50 (fine grade), and CNBr-activated Sepharose 4B were obtained from Pharmacia, Uppsala, Sweden. DEAE-cellulose (DE52) was obtained from Whatman. Mr standards and BSA were Sigma products. Analytical reagents were obtained from the following commercial sources: urease (type II), peroxidase (type II; RZ, 3.13), and L-glutamate dehydrogenase (type II) from Toyobo Co., Osaka, Japan; NADPH from Kohjin Co., Tokyo, Japan; restriction endonucleases and other enzymes from Takara Shuzo, Kyoto, or Toyobo, Osaka, Japan. SDS, isopropyl /J-D-thio-
galactopyranoside (IPTG), disuccinimidyl suberate, dimethyl sulphoxide and other chemical reagents were products of Nacalai Tesque Co., Kyoto, Japan. Oligonucleotides were synthesized
Abbreviation used: IPTG, isopropyl fl-D-thiogalactopyranoside. 1 Present address and address for correspondence and reprint requests: National Institute of Health and Nutrition, Shinjuku-ku, Tokyo 162, Japan. T Deceased.
Vol. 270
698
M. Ikemoto and others
with a DNA synthesizer (model 381A; Applied Biosystems) and purified by electrophoresis. Plasmid PTTQ18 (Stark, 1987) was purchased from Amersham International. E. coli K-12, strain HBI0I, which was obtained from Takara Shuzo Co., was used for bacterial transformation to construct plasmids. E. coli K- 12, strain KY1436 (Yano et al., 1987), was used for the expression of cloned cDNA for human liver arginase.
1984) was cloned into the BamHI site of pUC9 (Vieira & Messing, 1982). The resulting plasmid, pUCtacP, was digested by EcoRI, and the 200 bp fragment, containing a tac promotor, was ligated with pTAAl 1 digested by EcoRI. Finally plasmid pTAA12, containing tandem tac promotors, was obtained as shown at the bottom of Fig. 1. Expression of the arginase gene in E. coli E. coli K-12 strain KY1436 cells containing pTAA12 were grown in 10 litres of L broth containing ampicillin (50 jug/ml) at 30 'C. At an A600 of 1, 1 mM-IPTG was added. The bacteria were collected when the A600 was 3.5. A portion of the cells was resuspended in an adequate volume of 10 mM-Tris/HCl buffer, pH 7.4. The suspended cells were sonicated and centrifuged. The supernatant was used for the purification of arginase.
Construction of expression plasmids cDNA phARG6 for human liver arginase (Haraguchi et al., 1987) was used. Its 1.2 kb Sacl-Hindlll fragment, a synthetic double-strand oligonucleotide, and PTTQ18 (Stark, 1987) digested by EcoRI and HindIII were ligated as shown in Fig. 1. The resulting plasmid, pTAA 1I, was used for further construction. The 200 bp BamHI fragment of pKK223-3 (Brosius,
Eco R I site 8 Amino acid residues-
1-
..
BamHI
Hindill
Sacl EcoRI
Hindlil
Ne
EcoRl
I\ Sacl
Hindll
(
rrn Btl t2
pTTQ18
/cq
4 EcoRI Ptac
I,
EcoRI Sacl
Ptac, pTAA11 \Th
697
Expression of human liver arginase in Escherichia coli Purification and properties of the product Masaki IKEMOTO,* Masayoshi TABATA,* Toshio MIYAKE,t Toshiaki KONO,t Masataka MORI,4 Masayuki TOTANI§II and Takashi MURACHI§T *College of Medical Technology, Kyoto University, Kyoto 606, tBiotechnology Research Laboratory, TOSOH Corp., Ayase, Kanagawa-ken 252, llnstitute for Medical Genetics, Kumamoto University Medical School, Kumamoto 862, and §Department of Clinical Science and Laboratory Medicine, Kyoto University Faculty of Medicine, Kyoto 606, Japan
Arginase is an enzyme that catalyses the hydrolysis of arginine to urea and ornithine. It is abundantly present in the liver of ureotelic animals (i.e. those whose excretion is characterized by the excretion of uric acid as the chief end-product of nitrogen metabolism), but its purification has hitherto not been simple, and the yield not high. Starting with a partially truncated cDNA for human liver arginase recently made available, we constructed an expression plasmid that had tandemly linked tac promotors placed upstream of a full-length cDNA. By selecting Escherichia coli strain KY1436 as the host micro-organism, we established an efficient system for the production of human liver arginase protein. Chromatographies on CM-Sephadex G-150, DEAE-cellulose and Sephadex G-150, followed by preparative agar-gel electrophoresis, yielded 10 mg of apparently homogeneous enzyme protein from 1 g (wet wt.) of E. coli cells. E. coliexpressed human liver arginase had chemical, immunological and most catalytic properties indistinguishable from those of purified human erythrocyte arginase. However, E. coli-expressed arginase was a monomer of Mr 35000, whereas the purified erythrocyte arginase was trimer of Mr 105 000. They differed also in pH- and temperature-stabilities. Gel-filtration experiments with these two purified arginases under various conditions, as well as with unfractionated human liver and erythrocyte cytosol preparations, indicated that the native form of human arginase should be of Mr 35000, and that the trimeric appearance of human erythrocyte arginase after purification was an artifact of the purification procedures. It was thus concluded that, in Nature, the liver and erythrocyte arginases are identical proteins.
INTRODUCTION Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the final step of urea formation in the mammalian liver. Despite its abundance in the liver, purification of human liver arginase has been reported only with rather poor yield and purity (Bascur et al., 1966; Beruter et al., 1978) or with complicated methods (Greenberg et al., 1956; Beruter et al., 1978). Human erythrocytes have been used as a better starting material for the purification of human arginase, yielding an apparently homogeneous preparation of arginase, though again by rather tedious procedures (Nishibe, 1973). Moreover, no conclusive evidence has been obtained for the identity of this purified erythrocyte arginase with liver arginase. In addition, a fairly large amount of human liver arginase as a pure protein is needed, not only for biochemical studies, but also for more practical purposes. For example, the release of liver arginase into plasma has been reported to be useful as a diagnostic index of liver damage, such as that caused by carbon tetrachloride (Kaiju, 1938; Utzino & Ogawa, 1957; Ogawa, 1958). For developing an appropriate immunoassay system for laboratory diagnosis, pure antigen arginase is a prerequisite. Also, several cases of hyperargininaemia have been reported to reflect a hereditary deficiency of liver arginase (Cederbaum & Shaw, 1977; Spector et al., 1983; Bernar et al., 1986); in such cases human liver arginase may find a potential therapeutic application. Molecular cloning and nucleotide sequence of cDNA for human liver arginase has been reported, but the longest cDNA used for sequencing still lacked 24 nucleotides at its 5' end, thus yielding, when expressed in Escherichia coli, an arginase-like
product that had only 0.0180% of the enzymic activity of the native enzyme (Haraguchi et al., 1987). We therefore attempted to construct an expression plasmid housing a full-length cDNA for human liver arginase, and established a system for producing arginase protein that yielded as much as 10 mg of purified arginase per 1 g wet weight E. coli cells. The product, after purification by relatively simple procedures, was characterized in comparison with the human erythrocyte arginase that we had purified earlier (Ikemoto et al., 1989). Such a comparison led us to conclude that human liver and erythrocyte arginases are identical proteins, both having Mr values of 35 000 in their native states.
MATERIALS AND METHODS Materials CM Sephadex C-50, Sephadex G-150, Sephadex G-50 (fine grade), and CNBr-activated Sepharose 4B were obtained from Pharmacia, Uppsala, Sweden. DEAE-cellulose (DE52) was obtained from Whatman. Mr standards and BSA were Sigma products. Analytical reagents were obtained from the following commercial sources: urease (type II), peroxidase (type II; RZ, 3.13), and L-glutamate dehydrogenase (type II) from Toyobo Co., Osaka, Japan; NADPH from Kohjin Co., Tokyo, Japan; restriction endonucleases and other enzymes from Takara Shuzo, Kyoto, or Toyobo, Osaka, Japan. SDS, isopropyl /J-D-thio-
galactopyranoside (IPTG), disuccinimidyl suberate, dimethyl sulphoxide and other chemical reagents were products of Nacalai Tesque Co., Kyoto, Japan. Oligonucleotides were synthesized
Abbreviation used: IPTG, isopropyl fl-D-thiogalactopyranoside. 1 Present address and address for correspondence and reprint requests: National Institute of Health and Nutrition, Shinjuku-ku, Tokyo 162, Japan. T Deceased.
Vol. 270
698
M. Ikemoto and others
with a DNA synthesizer (model 381A; Applied Biosystems) and purified by electrophoresis. Plasmid PTTQ18 (Stark, 1987) was purchased from Amersham International. E. coli K-12, strain HBI0I, which was obtained from Takara Shuzo Co., was used for bacterial transformation to construct plasmids. E. coli K- 12, strain KY1436 (Yano et al., 1987), was used for the expression of cloned cDNA for human liver arginase.
1984) was cloned into the BamHI site of pUC9 (Vieira & Messing, 1982). The resulting plasmid, pUCtacP, was digested by EcoRI, and the 200 bp fragment, containing a tac promotor, was ligated with pTAAl 1 digested by EcoRI. Finally plasmid pTAA12, containing tandem tac promotors, was obtained as shown at the bottom of Fig. 1. Expression of the arginase gene in E. coli E. coli K-12 strain KY1436 cells containing pTAA12 were grown in 10 litres of L broth containing ampicillin (50 jug/ml) at 30 'C. At an A600 of 1, 1 mM-IPTG was added. The bacteria were collected when the A600 was 3.5. A portion of the cells was resuspended in an adequate volume of 10 mM-Tris/HCl buffer, pH 7.4. The suspended cells were sonicated and centrifuged. The supernatant was used for the purification of arginase.
Construction of expression plasmids cDNA phARG6 for human liver arginase (Haraguchi et al., 1987) was used. Its 1.2 kb Sacl-Hindlll fragment, a synthetic double-strand oligonucleotide, and PTTQ18 (Stark, 1987) digested by EcoRI and HindIII were ligated as shown in Fig. 1. The resulting plasmid, pTAA 1I, was used for further construction. The 200 bp BamHI fragment of pKK223-3 (Brosius,
Eco R I site 8 Amino acid residues-
1-
..
BamHI
Hindill
Sacl EcoRI
Hindlil
Ne
EcoRl
I\ Sacl
Hindll
(
rrn Btl t2
pTTQ18
/cq
4 EcoRI Ptac
I,
EcoRI Sacl
Ptac, pTAA11 \Th
