By a Cloned and Over-Expressed Gene in Escherichia Coli
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Biochem. J. (1987) 245, 875-880 (Printed in Great Britain) 875 Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli Nigel S. SCRUTTON, Alan BERRY and Richard N. PERHAM* Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 IQW, U.K. An expression vector, pKGR, for the gor gene from Escherichia coli encoding glutathione reductase was constructed by subcloning of an Avall fragment of the Clarke & Carbon bank plasmid pGR [Greer & Perham (1986) Biochemistry 25, 2736-2742] into the plasmid pKK223-3. The expression of glutathione reductase from the plasmid pKGR was found to have been successfully placed under the control of the tac promoter. Transformation of E. coli cells with this plasmid resulted in 100-200-fold increase in glutathione reductase activity in cell-free extracts. A rapid purification procedure for the enzyme, based on affinity chromatography on Procion Red HE-7B-CL-Sepharose 4B, was developed. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-gel electrophoresis, and all its properties were consistent with the DNA sequence of the gene [Greer & Perham (1986) Biochemistry 25, 2736-2742] and with those previously reported for E. coli glutathione reductase [Mata, Pinto & Lopez-Barea (1984) Z. Naturforsch. C. Biosci. 39, 908-915]. These experiments have enabled an investigation of the protein chemical and mechanistic properties of the enzyme by site-directed mutagenesis. INTRODUCTION from X-ray-crystallographic analysis of the protein at 0.2 nm resolution (Thieme et al., 1981; Pai & Schulz, Glutathione reductase (EC 1.6.4.2) catalyses the 1983). reduction of GSSG by NADPH: Human and E. coli glutathione reductases are highly GSSG + NADPH + H+ - NADP+ + 2GSH homologous in their amino acid sequences (Greer & It is a widespread member of an important family of Perham, 1986), making it certain that their three- flavoprotein oxidoreductases that includes dihydro- dimensional structures will also be similar. Thus it should lipoamide dehydrogenase (EC 1.6.4.3) (Reed, 1974; now be possible to test some of the predictions made Packman & Perham, 1982), thioredoxin reductase (EC about the catalytic mechanism of glutathione reductase 1.6.4.5) (Holmgren, 1980) and mercuric reductase (Fox (Pai & Schulz, 1983) by site-directed mutagenesis of the & Walsh, 1982). All these enzymes are dimers with an Mr E. coli protein. Complementary work is being undertaken of about 105000, and all possess a disulphide bridge in with mercuric reductase (Schultz et al., 1985). To each subunit which is alternately oxidized and reduced facilitate such a study, it is essential to have a system for as part of the catalytic mechanism (reviewed by expression ofthe cloned gene, preferably in a controllable Williams, 1976). Amino acid sequences around the expression vector, and a simple method for purifying the disulphide bridges in dihydrolipoamide dehydrogenase, enzyme from transformed cells. We describe here the glutathione reductase and mercuric reductase are highly cloning of the E. coli gor gene into a plasmid vector-that homologous, implying that they have arisen by divergent raises the glutathione reductase activity some 200-fold evolution from a common ancestor (Perham et al., 1978; above that found in untransformed wild-type E. coli Williams et al., 1982; Fox & Walsh, 1983). On the other (strain JM101) and a simple purification method that hand, thioredoxin reductase is sufficiently different for it permits the preparation of large quantities of E. coli to be likely that this enzyme has arisen by convergent glutathione reductase indistinguishable from that des- evolution towards a common mechanism (Perham et al., cribed by Mata et al. (1984). 1978). The lpd gene of Escherichia coli, encoding dihydro- MATERIALS AND METHODS lipoamide dehydrogenase (Stephens et al., 1983), the merA gene of transposon Tn501 from Pseudomonas Materials aeruginosa, encoding mercuric reductase (Brown et al., Complex bacteriological media were from Difco 1983), and the gor gene of E. coli, encoding glutathione Laboratories and all media were prepared as described in reductase (Greer & Perham, 1986), have been cloned and Maniatis et al. (1982). L-[35S]Methionine (800 Ci/mmol; their nucleotide sequences determined. This in turn has for coupled transcription-translation) and [a-[35S]- enabled their complete primary structures to be inferred thio]dATP triethylammonium salt (> 400 Ci/mmol; for and compared. The complete amino acid sequence of DNA sequencing) were from Amersham International. human glutathione reductase (Krauth-Siegel et al., 1982) Ethidium bromide, isopropyl ,-thiogalactoside, 5- has also been determined, which has permitted con- bromo-4-chloroindol-3-yl ,-galactoside, NADPH, siderable light to be thrown on the reaction mechanism GSSG and amino acids were from Sigma Chemical Co. * To whom correspondence should be addressed. Vol. 245 876 N. S. Scrutton, A. Berry and R. N. Perham Deoxy- and dideoxy-nucleoside 5'-triphosphates used in until the required cell density was reached. For DNA sequencing were from P-L Biochemicals. Ultrapure small-scale preparation ofextract, a 10 ml culture ofcells agarose, dithiothreitol and CsCl were from Bethesda was grown and then centrifuged at 300 g for 5 min. The Research Laboratories. Procion Red HE-7B linked to medium was decanted and the pellet washed in 2 ml of CL-Sepharose 4B was a gift from Dr. C. R. Lowe sonication buffer (50 mM-potassium phosphate, pH 8.0). (Biotechnology Centre, University of Cambridge). All Cells were again collected by centrifugation (3000 g for other chemicals were ofanalytical-reagent grade wherever 5 min) and resuspended in 1 ml of sonication buffer. The possible. Glass-distilled water was used throughout. suspension was then sonicated in three pulses each of 8 s The restriction enzymes HindIII, Avall, EcoRI and on power setting 2 on a Heat Systems cell disruptor. The SmaI were purchased from New England Biolabora- cell extract was centrifuged (11 500 g for 5 min) to yield tories. Calf intestinal alkaline phosphatase was obtained a cell-free supernatant. Protein concentration was from Boehringer Mannheim. T4 DNA polymerase was determined either by the method of Lowry et al. (1951) from Pharmacia. T4 DNA ligase and the Klenow or by that of Bradford (1976); bovine serum abumin was fragment of E. coli DNA polymerase I were generously used as standard. made available by Dr. R. T. Hunt (Department of Biochemistry, University of Cambridge). The expression Assay of glutahione reductase vector pKK223-3 was from Pharmacia. Glutathione reductase activity was estimated at 30 °C by the GSSG-dependent oxidation ofNADPH measured Plasmid construction and DNA sequencing by the decrease in the absorbance at 340 nm (Mata et al., Plasmid or bacteriophage RF DNA were prepared on 1984). Each assay mixture contained 10 1 of 1O mM- a 100 ml scale as described by Maniatis et al. (1982). For NADPH, 10 1 of 120 mM-GSSG, the sample to be the purposes of screening, plasmids and RF DNA were assayed (10-100,ul) and 0.1 M-potassium phosphate prepared on a small scale by the rapid boiling technique buffer, pH 7.6, to a final volume of 1 ml. of Holmes & Quigley (1981). Endonuclease digestion of Amino acid composition and sequence analysis DNA was carried out as recommended by the enzyme suppliers. For analytical digests 1,ug of DNA was Amino acid analysis was carried out on an LKB 4400 overdigested ten times for 1 h in a total volume of 25 4u1. amino acid analyser. The samples were hydrolysed for For preparative digests, 100 ,ug ofDNA was overdigested 24, 48 and 96 h in 6 M-HCI, as described by Perham ten times in a total volume of 400,l overnight. DNA (1978). N-Terminal sequence analysis was kindly carried fragments were purified by submarine agarose-gel out by Applied Biosystems on a gas-phase sequencer. electrophoresis. The gels were electrophoresed in TAE Determination of native value buffer (40 mM-Tris/20 mM-sodium acetate/lO mM- Mr EDTA, pH 8.2) at 5V/cm. Nucleic acid was stained with The Mr of E. coli glutathione reductase was ethidium bromide in running buffer (0.5 jig/ml) for determined by gel-filtration fast protein liquid chroma- 20 min, and DNA was detected by using a long- tography on a Pharmacia Superose 12 column in wavelength u.v. transilluminator. The appropriate gel 50 mM-potassium phosphate buffer, pH 7.5. The follow- band in the stained agarose gel was excised and placed ing proteins were used as standards (numbers in in an ISCO gel electroelution cup and electroeluted in parentheses indicate the assumed Mr values): ferritin accordance with Allington et al. (1978). The eluted DNA (480000), rabbit fructose-bisphosphate aldolase was purified further by phenol, chloroform and diethyl (160000), calf alkaline phosphatase (140000), bovine ether extraction (Maniatis et al., 1982). serum albumin (67500), chicken ovalbumin (44000), Vector DNA was cut with the appropriate enzyme, carbonic anhydrase (32000), a-chymotrypsinogen A treated with calf intestinal alkaline phosphatase if (25000), horse heart myoglobin (18800), hen egg-white required and then ethanol-precipitated (Maniatis et al., lysozyme (14388) and ox insulin (11466). 1982). End-repair of DNA with T4 polymerase, ligation of DNA fragments and transformation ofcells were also RESULTS AND DISCUSSION carried out as described by Maniatis et al. (1982). Bacteriophage single-stranded DNA was prepared and Construction of an expression vector for glutathione sequenced by the chain-termination method (Sanger reductase et al., 1980; Biggin et al., 1983). Insertion of the gor gene into a suitable M13 bacteriophage would not only provide a convenient Coupled transcription-translation of DNA in vitro source of single-stranded DNA for site-directed muta- genesis experiments, but the possibility existed that the Coupled transcription-translation ofDNA was carried high copy number of the recombinant bacteriophage out as described by Zubay (1974) with a cell-free system DNA within the host cell might cause a desirable derived from E.