High-Level Expression of Enzymatically Active Human Cu/Zn Superoxide Dismutase in Escherichia Coli (Bacterial Expression Vector/A PL/Ribosome Binding Site) JACOB R
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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 7142-7146, October 1986 Biochemistry High-level expression of enzymatically active human Cu/Zn superoxide dismutase in Escherichia coli (bacterial expression vector/A PL/ribosome binding site) JACOB R. HARTMAN, TANIA GELLER, ZIVA YAVIN, DANIEL BARTFELD, Dov KANNER, HAIM Aviv, AND MARIAN GORECKI Bio-Technology General (Israel) Ltd., Kiryat Weizmann, Rehovot 76326 Israel Communicated by Richard Axel, May 13, 1986 ABSTRACT Expression of human Cu/Zn superoxide dis- interest has evolved in the therapeutic potential of SOD. A mutase (SOD) with activity comparable to the human eryth- wide range of clinical applications has been suggested. These rocyte enzyme was achieved in Escherichwa coli by using a include prevention of oncogenesis and tumor promotion, vector containing a thermoinducible X PL promoter and a reduction of the cytotoxic and cardiotoxic effects of ,8-lactamase-derived ribosomosal binding site. The recombi- anticancer drugs (5), anti-inflammatory action (6), and pro- nant human SOD was found in the cytosol ofdisrupted bacteria tection against reperfusion damage of ischemic tissues (7). In and represented >10% of the total bacterial protein. The addition, there is much interest in studying the effects ofSOD enzyme was purified to homogeneity by salt precipitation, gel on the aging process (8). filtration chromatography, and ion exchange chromatography. The exploration ofthe therapeutic potential ofhuman SOD The active enzyme was obtained in high yield only when 1 mol has been hindered by its limited availability. The enzyme is of copper and 1 mol of zinc were incorporated into each mol of a dimeric metalloprotein composed of identical noncovalent- subunit during bacterial growth or by reconstitution of the ly linked subunits, each of 16 kDa and containing one atom apoenzyme. Human Cu/Zn SOD produced in bacteria has an ofcopper and one atom ofzinc (9). Each subunit is composed apparent subunit molecular massof19 kDaon NaDodSO4/poly- of 153 amino acids of known sequence (10, 11). Recently, a acrylamide gels. The native enzyme behaves as a dimer of 32 cDNA clone containing the entire coding region of human kDa as determined by gel filtration. Sequence analysis of the SOD was isolated and sequenced (12, 13). The gene coding NH2 terminus revealed that the first 14 amino acids corre- for human SOD was introduced by us into an efficient sponded to authentic human SOD except that the NH2-terminal bacterial expression vector. We report here the production of alanine was not acetylated. Thus, the bacterial processing gram quantities of enzymatically active human Cu/Zn SOD system readily removes the NH2-terminal methionine residue in Escherichia coli. from recombinant human SOD. MATERIALS AND METHODS The possible biological role of superoxide dismutase (SOD; Bacterial Growth and Induction Protocol. Construction of superoxide:superoxide oxidoreductase, EC 1.15.1.1) as an human SOD-expressing plasmid pSOD/31Tll was carried out oxygen free radical (O2j) scavenger was proposed in 1968 by according to published procedures (14) and will be described McCord and Fridovich (1) and has provoked considerable in detail elsewhere. Plasmid pSODJ31T11 was propagated in interest within the scientific community since that time. E. coli strain A1645 [c600 r- m+ gal' thr- leu- lac- bl (X Superoxide radicals and other highly reactive oxygen species cI857AHJ ABamHI N+)] (15), which produces X cI857 are produced in every respiring cell as by-products of repressor and the N gene product. Overnight cultures were oxidative metabolism, and they have been shown to cause grown at 30°C in L broth supplemented with tetracycline extensive damage to a wide variety of macromolecules and (12.5 ,ug/ml) and used to inoculate a 50-liter fermentor cellular components (for reviews, see refs. 2 and 3). A group containing casein hydrolysate (20 g/liter), yeast extract (10 of metalloproteins known as superoxide dismutases catalyze g/liter), glucose (10 g/liter), K2HPO4 (2.5 g/liter), the oxidation-reduction reaction MgSO4-7H2O (1 g/liter), NaCl (5 g/liter), and tetracycline (12.5 mg/liter). The pH was maintained at 7.0 ± 0.2 with 20j + 2H+ -- H202 + 02 NH3. Induction was started when cell concentration reached an OD6w of =10.0. At that stage, an additional 10g ofglucose and thus provide a defense against oxygen toxicity. There are per liter was added, the temperature was raised, and the three known forms of SOD that contain different metals- fermentation was allowed to continue at 42°C for 75 min. namely, iron, manganese, or both copper and zinc. All of Analysis of Bacterial Extracts. Bacterial cells were harvest- these catalyze the same reaction with high efficiency, and all ed by centrifugation and suspended in 50 mM potassium operate by a similar mechanism in which the metal is the phosphate buffer (pH 7.8). Aliquots were lysed in 3 x sample catalytic factor in the active site. These enzymes fall into buffer [30% (vol/vol) glycerol/9% NaDodSO4/2.1 M 2-mer- several evolutionary groups. The Fe-containing SODs are captoethanol/187.5 mM Tris HCl, pH 6.8/0.5% bromo- found primarily in prokaryotic cells, while Cu/Zn SODs have phenol blue), heated at 100°C for 5 min, and proteins were been demonstrated in all higher eukaryotes. Mn SODs exist analyzed on 15% NaDodSO4/polyacrylamide gels (16, 17). throughout the phylogenetic range, from microorganisms to Proteins separated on polyacrylamide gels were electropho- humans (reviewed in ref. 4). retically transferred to nitrocellulose sheets (18) (0.45 ,um; Since every biological macromolecule can serve as a target Schleicher & Schuell) and underwent reaction with rabbit for the damaging action of the abundant oxygen radical, anti-human SOD IgG (affinity-purified from rabbit antisera raised against commercial human erythrocyte SOD; Sigma) The publication costs of this article were defrayed in part by page charge followed by incubation with 1251I-labeled protein A. The blots payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: SOD, superoxide dismutase; bp, base pair(s). 7142 Downloaded by guest on September 27, 2021 Biochemistry: Hartman et A Proc. Natl. Acad. Sci. USA 83 (1986) 7143 were thoroughly washed, air-dried, and exposed to Agfa- Gevaert curix RP2 x-ray film. Purification of the Recombinant Human SOD. Purification ofthe enzyme was accomplished by the following procedure: Induced bacteria were suspended in 50 mM potassium phosphate (pH 7.8) and disrupted by sonication. The lysate was centrifuged and the clear supernatant was passed through a cushion of DEAE-cellulose to remove nucleic acids. The eluate was treated with 2% NaDodSO4 at 370C for 1 hr. This procedure was shown to inactivate eukaryotic Mn SOD (19), and we found that it also inactivates the bacterial SOD activities (unpublished observations). Excess of NaDodSO4 was then removed by addition of potassium chloride to a final concentration of 0.3 M. The precipitate containing KDodSO4 was centrifuged out and the solution was treated with ammonium sulfate to 60% saturation. The precipitated proteins were removed by centrifugation. The supernatant was dialyzed against 20 mM potassium phos- phate (pH 7.8), concentrated by ultrafiltration, and separated by gel chromatography on a Fractogel 55 column. Fractions containing human SOD were identified by gel electrophore- sis, pooled, dialyzed, and brought to 2 mM potassium FIG. 1. Schematic description of clone pSOD,31T11. Construc- phosphate (pH 7.8) and loaded on a DE-52 column, which tion ofthe clone expressing human SOD (hSOD) is described in detail was developed with a linear gradient of 2-150 mM potassium elsewhere. Plasmid pSOD,/1T11 is 3700 bp long, contains the X PLOL phosphate (pH 7.8). The human SOD peak was collected and region, including the nutL site, the P-lactamase gene (of pBR322) dialyzed, and the ionic strength was adjusted to 2 mM promoter and ribosome binding site, the entire human SOD coding potassium phosphate (pH 7.8) and then subjected to a second sequences, and confers resistance to tetracycline (see text). The DE-52 column. Homogeneous human SOD was eluted with DNA sequence at the junction between the 5' end of the SOD gene 15 mM potassium phosphate (pH 7.8). and the ribosomal binding site derived from 3-lactamase are pre- sented with the two possible translation start codons underlined. The Analysis of Induced Human SOD. SOD activity was mea- ribosome binding site is indicated with a dotted line under the sured by monitoring the inhibition of reduction of fer- nucleotide sequence. Five amino acids corresponding to the amino ricytochrome c, as described by McCord and Fridovich (1). terminus of authentic human SOD are also shown. Bacterial and human SOD activities were differentiated by using 1 mM KCN (20). Protein concentration was determined by the method of Lowry et al. (21) using bovine serum 1). Construction and cloning of the various expression albumin as a standard. Cu and Zn content in homogeneous vectors that resulted in the generation of plasmid SOD preparations were determined by atomic absorption. pSOD,31T11 will be described in detail elsewhere. SOD apoenzyme was prepared according to Weser and Expression of Human Cu/Zn SOD in E. coli. Plasmid Hartmann (22). Reconstitution was performed by simulta- pSODB1T11 was propagated in anE. coli strain that produces neous addition of Cu2+ and Zn2+ to highly purified constitutively the thermolabile repressor cI857 (25) and the apoenzyme (23). Human Cu/Zn SOD (Sigma) and bovine transcription antiterminator Ngene product (26). At 30°C, the Cu/Zn SOD (Grunenthal, Aachen, F.R.G.) were used as repressor binds to OL and blocks transcription from the standards. strong PL promoter. Nevertheless, a low level of expression Amino Acid Sequencing. NH2-terminal amino acid se- of human SOD is detected by immunoblot analysis (Fig.