©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Purification, Cloning, and High Level Expression of a Glutaredoxin-like Protein from the Hyperthermophilic Archaeon Pyrococcus furiosus (*)

(Received for publication, November 28, 1994)

Annamaria Guagliardi (1)(§) Donatella de Pascale (1) Raffaele Cannio (1) Valentina Nobile (1) Simonetta Bartolucci (1) Mosè Rossi (1) (2)

From the  (1)Dipartimento di Chimica Organica e Biologica, Università di Napoli, Via Mezzocannone 16, Napoli 80134 and (2)Istituto di Biochimica delle Proteine ed Enzimologia, C.N.R., Via Marconi 10, Napoli 80125, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A protein has been purified to homogeneity from crude extracts of the hyperthermophilic archaeon Pyrococcus furiosus based on its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol; the protein has a molecular mass of 24.8 kDa and a pI of 4.9, and it is highly heat-stable. The first 29 amino acid residues at the N terminus of the P. furiosus protein were determined by Edman degradation, and its gene was cloned in Escherichia coli. The amino acid sequence derived from the DNA sequence contains the CPYC sequence, which is typical of the active site of glutaredoxin (also called thioltransferase). The C-terminal portion of the P. furiosus protein, containing the conserved sequence, shows sequence similarity with glutaredoxins from different sources. The P. furiosus protein can reduce disulfide bonds in L-cystine in the presence of GSH (the thioltransferase activity) with an optimum pH of 8.0. The expression of the P. furiosus protein, with full activity, in E. coli at a very high level (21% of total soluble protein) is described; the recombinant protein was purified to homogeneity by merely two successive heat treatments and gel filtration chromatography. The features of the P. furiosus protein here described are discussed in light of the current knowledge about the ubiquitous family of protein disulfide oxidoreductases.


INTRODUCTION

The ubiquitous family of protein disulfide oxidoreductases comprises proteins whose cysteine residues in the active site sequence CXXC undergo reversible oxidation-reduction. The protein disulfide isomerase in eucaryotic endoplasmic reticulum, thioredoxin, and glutaredoxin (also known as thioltransferase) are the most thoroughly studied members of the family. Protein disulfide isomerase (a dimer of 57 kDa) has the sequence CGHC in the active site; the protein disulfide isomerase homologue in Escherichia coli, named DsbA, is a monomer of 189 residues with the active sequence CPHC. Protein disulfide isomerase and DsbA catalyze the reduction, oxidation, and reshuffling of protein disulfides depending on the redox potential, thus playing a primary role in the correct structure and, hence, in the proper functioning of protein molecules (reviewed by Freedman(1992), Bardwell and Beckwith(1993), and Loferer and Hennecke(1994)). Thioredoxin, similar to glutaredoxin in molecular mass (about 12 kDa), has at the N terminus the active sequence CGPC, with the only exception represented by the thioredoxin C2 of Corynebacterium nephridii where A replaces G (reviewed by Holmgren(1985) and Eklund et al.(1991)). Glutaredoxin has at the N terminus a redox disulfide bond in the sequence CPYC, with the exceptions of glutaredoxins from T4 (CVYC), pig liver, and rice (CPFC) (reviewed by Holmgren, 1989). Thioredoxin and glutaredoxin have been postulated to be involved in a number of different cellular processes, as cofactors for dithiol-disulfide exchange reactions on small and protein disulfides.

All living beings can be grouped into three evolutionarily distinct kingdoms: Eucarya, Bacteria, and Archaea (Woese et al., 1990). All but two genera of hyperthermophiles, the microorganisms that grow at and above 100 °C, belong to the kingdom of Archaea. Eucarya and Archaea have a common ancestor, which is not shared by Bacteria; the first organisms to have diverged from the Eucarya/Archaea lineage were the hyperthermophiles, considered to be the most ancient of all extant life. Pyrococcus furiosus, the best studied among the hyperthermophiles that reduce elemental sulfur (S°) to H(2)S, is a strictly anaerobic heterotroph; S° is reduced probably to remove toxic H(2), not to gain the energy for growth that is obtained by an unusual fermentative type metabolism (Adams, 1994). The enzymes and the proteins so far isolated from P. furiosus exhibit unique features with respect to their non-archaeal counterparts (for a review see Adams, 1993).

This paper reports the isolation from P. furiosus of a novel 24.8-kDa protein disulfide oxidoreductase, which exhibits glutaredoxin-like properties. The cloning and the high level expression of the coding gene in E. coli are also described.


EXPERIMENTAL PROCEDURES

Materials

Bovine insulin, GSH, NADPH, glutathione reductase from yeast (190 units/mg), and molecular weight standards for SDS-PAGE (^1)were obtained from Sigma. Ampholynes and pTRC99A plasmid were from Pharmacia Biotech Inc. Radioactive materials were obtained from Amersham Corp. Plasmid pGEM7Zf(+), deoxynucleotides, and restriction and modification enzymes were purchased from Promega Biotec. Plasmid pUC18 was from Boehringer Mannheim. E. coli strains Rb791 and BO3310 were kindly provided by Prof. G. Sannia (Dipartimento di Chimica Organica e Biologica, Università di Napoli, Italy) and Prof. F. Blasi (Dipartimento di Genetica, Università di Milano, Italy), respectively. All materials used for gene amplification were supplied by Stratagene Cloning Systems. All synthetic oligonucleotides were purchased from Primm. All other chemicals were of the highest grade available.

Analytical Methods for Protein and DNA

Protein concentration was determined by the Bio-Rad dye-binding assay (Bradford, 1976), using bovine serum albumin as the standard. SDS-PAGE was performed according to Laemmli(1970); samples were heated at 100 °C for 30 min in 2% SDS, 5% 2-mercaptoethanol, and the run was performed using 5% stacking gel and 12.5% separating gel. Isoelectric focusing was carried out on 5% polyacrylamide gel at 4 °C using 2% ampholytes in the pH range 2.5-7; isoelectric point was estimated using a surface electrode (Righetti and Drysdale, 1976). N-terminal sequence analysis was performed by Edman degradation on an Applied Biosystem 473A sequencer according to the manufacturer's instructions.

DNA electrophoresis on 1% agarose gels in 90 mM Tris borate, 20 mM EDTA (TBE buffer) and plasmid transformations of E. coli cells were carried out as described by Sambrook et al.(1989). DNA sequencing was carried out by the dideoxy chain termination method (Sanger et al., 1977) with [S]dATP, using the Sequenase version 2.0 sequencing kit (USB) on alkali-denatured double-stranded templates; the universal M13 forward and reverse primers or specific synthetic oligonucleotides were used.

Activity Assays

The insulin reductase activity was assayed according to Holmgren(1979). The standard assay mixture contained 0.1 M sodium phosphate buffer, pH 7.0, 0.13 mM bovine insulin in the absence or in the presence of the P. furiosus protein (final volume, 1 ml); upon the addition of 1 mM DTT, the increase of the absorbance at 650 nm was monitored at 30 °C.

The standard assay mixture for the thioltransferase activity consisted of 0.1 M sodium phosphate buffer, pH 8.0, 1 mM EDTA, 10 mM GSH, 2.5 mML-cystine, 0.2 units of glutathione reductase, 0.3 mM NADPH in the absence or in the presence of the P. furiosus protein (final volume, 1 ml); the enzyme activity was monitored at 340 nm and 30 °C. The net enzymatic reaction rate, expressed as micromoles of NADP formed per minute, was obtained by subtraction of the nonenzymatic reaction rate from the total rate. The activity dependence on pH was determined by the standard assay mixture in which 0.1 M sodium phosphate was used from pH 6 to 8 and 0.1 M Tris-HCl from 8 to 9 (0.12 µM protein was used for each assay).

Protein Purification

All steps were performed at 4 °C, using buffers degassed and flushed with N(2). Samples were concentrated by ultrafiltration through a membrane of 2,000 Da cut-off using an Amicon cell. The insulin precipitation assay was used to monitor the protein activity.

Preparation of the Crude Extract

P. furiosus was grown at 95 °C and pH 7.5 in a medium containing the following (per liter): bacto peptone (5 g), yeast extract (1 g), NaCl (19.4 g), MgSO(4) (12.6 g), Na(2)SO(4) (3.2 g), CaCl(2) (2.4 g), KCl (0.5 g), NaHCO(3) (0.16 g), KBr (0.08 g), SrCl(2) (0.057 g), H(3)PO(3) (0.022 g), Na(2)SO(3) (0.004 g), NaF (0.0024 g), KNO(3) (0.0016 g), Na(2)HPO(4) (0.01 g), and sulfur (25 g). The culture was bubbled with N(2) (5 liters/min). Cells, harvested after 8 h, were immediately frozen in liquid N(2) and stored at -80 °C until required.

Ten grams of bacteria underwent freeze-thawing twice; were mixed with 30 g of sand and 100 ml of 20 mM Tris buffer, pH 8.4, 2 mM EDTA, 1 M NaCl; and were homogenized in a Homni Mixer. The homogenate was centrifuged at 4,000 times g for 20 min at 4 °C to remove the sand and ultracentrifuged at 160,000 times g for 90 min at 4 °C; the supernatant represented the crude extract.

Anion Exchange Chromatography

The crude extract (1.3 g) was extensively dialyzed against 20 mM Tris buffer, pH 8.4, 2 mM EDTA (Buffer A) and loaded onto a DEAE-Sepharose Fast Flow column (Pharmacia, 2.2 times 18 cm) equilibrated in Buffer A. Bound proteins were eluted at a flow rate of 50 ml/h by a linear gradient 0-0.3 M NaCl in Buffer A. The active fractions were pooled and concentrated.

First Gel Filtration Chromatography

The DEAE pool (40 mg) was loaded in three separate runs onto a HiLoad Superdex 75 column (Pharmacia, 2.6 times 60 cm) connected to an FPLC system (Pharmacia) eluted with 20 mM Tris buffer, pH 8.4, 2 mM EDTA, 1 M NaCl (Buffer B) at a flow rate of 2 ml/min. The active fractions were pooled and concentrated.

Second Gel Filtration Chromatography

The Superdex 75 pool (1.7 mg) was loaded onto a Bio-Gel P30 column (Bio-Rad, 1.6 times 70 cm) eluted with Buffer B at a flow rate of 6 ml/h. The active fractions were concentrated (0.5 mg) and stored at -20 °C.

Material for N-terminal amino acid sequence analysis was prepared by C4 reverse-phase HPLC.

Isolation of Chromosomal P. furiosus DNA

Ten grams of P. furiosus cells were suspended in 25 ml of 50 mM Tris-HCl buffer, pH 8.0, 10 mM EDTA (washing buffer) and centrifuged at 3000 times g. Cells were resuspended in 20 ml of washing buffer containing 0.2% Triton X-100 and 1% SDS; 10 strokes were applied to the samples in potter Dounce. The soluble fraction, cleared by centrifugation at 129,500 times g for 30 min at 4 °C, was heated for 10 min at 65 °C and again centrifuged. Cesium chloride (1 g/ml) and ethidium bromide (0.6 mg/ml) were added, and the samples were ultracentrifuged at 352,000 times g for 16 h at 16-18 °C. Chromosomal DNA bands were revealed by irradiation of a 340-nm UV lamp and withdrawn using a hypodermic needle as described by Sambrook et al.(1989). Ethidium bromide and cesium chloride were removed, respectively, by repeated extraction with isoamylic alcohol and extensive dialysis against 10 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA. DNA concentration was determined spectrophotometrically at 260 nm, and molecular weight was checked by electrophoresis on 0.6% agarose gel in TBE buffer using suitable DNA molecular size markers.

Construction of P. furiosus Gene Bank

High molecular weight DNA of P. furiosus was partially digested with Sau3AI (1 h of incubation with 0.15 units per µg of genomic DNA), and the fragments of 2-4-kb size were isolated by electroelution from 3.5% polyacrylamide gel electrophoresis. The fragments were subjected to partial filling in with E. coli DNA polymerase (Klenow fragment), dGTP, and dATP and inserted into the vector pGEM7Zf(+), previously made end-compatible by linearization with XhoI and partial filling in with the Klenow fragment, dCTP, and dTTP. E. coli BO3310-competent cells were transformed with the ligation mixture; the growth in Luria-Bertani medium containing 100 µg/ml ampicillin for 4 h (Sambrook et al., 1989) allowed the propagation and the amplification of the gene bank.

Southern Blot Analysis

Chromosomal DNA of P. furiosus was digested with restriction endonucleases, subjected to agarose gel electrophoresis and trasferred to a nylon membrane (Hybond N, Amersham). Ten pmol of 41-mer oligonucleotide mixture (5`-GATAAGAAGGT(G/T)AT(T/A)AAGGAGGAGTTTTTT(T/A)(C/G)(T/G)AAGATGGT-3`), designed on the basis of the N-terminal sequence of the protein, was end-labeled with [-P]ATP by T4 polynucleotide kinase; excess label was removed by passage through a Nensorb20 Nucleic Acid Purification Cartridge (DuPont NEN). The labeled probe (10^6 cpm/pmol) was incubated with the nylon membrane for 16 h at 42 °C in 10 ml of the hybridization mixture (5 times SSC, 0.5% SDS, 5 times Denhardt solution) according to Sambrook et al.(1989); the filter was washed with 5 times SSC, 0.5% SDS (two changes of 30 min each) at 45 °C and at 55 °C.

Cloning of the Gene

The screening of the genomic bank of P. furiosus in pGEM7Zf(+) was performed using colony hybridization experiments carried out under the conditions of stringency assessed for the Southern blot analysis. Replica filters were used as a control of signal specificity. Clones positive to a first selection were definitively isolated by a second colony hybridization screening performed according to the same protocol. The whole coding sequence was labeled by incorporation of [alpha-P]dATP using the random priming method and the specific kit purchased from Boehringer Mannheim. Suitable restriction fragments were subcloned into pUC18 plasmid and sequenced using the universal M13 forward and reverse primers; all the sequences were performed in duplicate on both strands using the specific synthetic oligonucleotides Pf1 (5`-AGGTTGACAAACCTAG-3`), Pf2 (5`-GAGCCATTCTAACGGC-3`), Pf3 (5`-CTCCAAAGTTCCTTTCCC-3`), and Pf4 (5`-GAGAGCTGAGAGTAAC-3`), which were designed on the basis of the sequence analysis described above.

Construction of the Expression Vector

The complete coding sequence of the gene was isolated by gene amplification performed according to Saiki(1990) for 25 cycles at 55 °C annealing temperature, on a Perkin Elmer Cycler Temp. The oligonucleotides used as primers were 5`-GcgccATGGGATTGATTAGTGAC-3` (PfN) and 5`-gagAGTCGAGAGTCGACTAGATCTGCG-3` (PfC), where underlined letters indicate the initiation and termination codons and small letters indicate point mutations inserted to generate the NcoI and XbaI sites; the pDR7 plasmid, containing the whole gene, was linearized with BamHI and used as template for Pf.U polymerase. The amplified fragment was digested with NcoI and XbaI and inserted in pTRC99A plasmid, previously made end-compatible with the same restriction endonucleases. Restriction analysis and subsequent sequencing using Pf1, Pf2, Pf3, and Pf4 oligonucleotides as primers confirmed the correct sequence of the recombinant clone, which we designated as pGlx.

High Level Expression and Purification of the Recombinant Protein

E. coli Rb791-competent cells were transformed with the pGlx expression vector and grown at 37 °C to different cell densities in 1 liter of Luria-Bertani medium; IPTG was added to 1 mM final concentration, and the induction time was varied from 2 to 24 h. Rb791 cells transformed with pTRC99A represented a negative control. Cells were harvested by centrifugation at 3,000 times g for 10 min at 4 °C, washed with 30 ml of ice-cold 50 mM sodium phosphate buffer, pH 8.0, 0.1 M NaCl, 1 mM EDTA, and resuspended in 48 ml of the same buffer supplemented with 0.7 mM phenylmethanesulfonyl fluoride. Cells were disrupted by 20-min incubation at room temperature in the presence of lysozyme (11 mM); after the addition of sodium deoxycholate (2.4 mM) and DNase I (10 nM), the suspension was incubated at room temperature for 30 min and then centrifuged at 5,000 times g for 15 min at 4 °C. Protamine sulfate (1 mg/ml final concentration) was added to precipitate nucleic acids, and the supernatant of a centrifugation at 5,000 times g for 15 min at 4 °C represented the crude extract.

The recombinant protein was purified to homogeneity, as judged from SDS-PAGE analysis, by the following procedure, starting from the crude extract of transformed cells grown at 1.0 A cell density and induced for 18 h with IPTG.

Heat Treatments

The crude extract (85 mg) was heated at 65 °C for 10 min and centrifuged at 5,000 times g at 4 °C for 10 min; the supernatant was concentrated by ultrafiltration in an Amicon cell (membrane cut-off 2,000 Da), subjected to a second heating at 70 °C for 10 min, and centrifuged as above.

Gel Filtration Chromatography

The sample from heat treatment (28 mg) was loaded onto a HiLoad Superdex 75 column (Pharmacia, 2.6 times 60 cm) connected to an FPLC system (Pharmacia) eluted with Buffer B at a flow rate of 2 ml/min. Pure protein (18 mg) was stored at -20 °C.


RESULTS

Purification and Physical Properties of the P. furiosus Protein

The reduced form of protein disulfide oxidoreductases is a powerful reductant of disulfide bridges in small molecules and proteins. The reduction of protein disulfides can be easily measured using insulin as substrate; upon reduction of the interchain disulfide bridges between chains A and B of insulin, the turbidity of the solution increases because of precipitation of free B chain (Holmgren, 1979). The cytosol of P. furiosus was found to catalyze the insulin reduction in the presence of DTT at 30 °C. The protein responsible for this activity was purified to homogeneity by one anion exchange and two gel filtration chromatographies; SDS-PAGE analysis of the active sample from the second gel filtration column showed one band whose molecular mass was 24.8 kDa (Fig. 1). The reductase activity was found to be associated with this protein throughout the purification steps; in other words, the protein that is isolated seems to be the only one able to effectively reduce insulin disulfide bonds under the assay conditions employed. Because the assay based on insulin precipitation is difficult to quantify, no yield and purification factor can be calculated. Pure protein can be stored at -20 °C for at least 3 months without loss of activity; the presence of a reducing agent does not seem to be essential for its stability.


Figure 1: SDS-PAGE analysis of the active sample during the purification procedure of the P. furiosus protein. Lane 1, pooled active fractions from DEAE-Sepharose column; lane 2, pooled active fractions from Superdex 75 column; lane 3, pooled active fractions from Bio-Gel P30 column; lane 4, molecular weight standards. Samples (6 µg/lane), treated and run as described under ``Experimental Procedures,'' were stained with Coomassie Blue.



Isoelectric focusing gel electrophoresis of the homogeneous protein revealed one band whose isoelectric point was 4.9.

The protein purified from P. furiosus was endowed with an extreme resistance toward heating: after 3 h of incubation at 90 °C, its effect on the insulin precipitation reaction was comparable with that of the native protein.

N-terminal Sequence Analysis, Cloning, and DNA Sequencing

The homogeneous protein from P. furiosus was subjected to automatic Edman degradation, and the following 29 residues were positively identified: GLISDADKKVIKEEFFSKMVNPVKLIVFV. Based on the amino acid sequence from residue 8 to residue 20 and the codon usage in Archaea (Hain et al., 1992), a 41-mer oligonucleotide mixture was designed.

The strategy used to establish a representative genomic bank in pGEM7Zf(+) plasmid (Fig. 2) was highly efficient for the insertion of one single DNA fragment from P. furiosus genome per vector molecule (Cannio et al., 1994). The specificity as a probe of the oligonucleotide mixture and the stringency conditions were previously determined by Southern blot analysis of the genomic DNA digested with different restriction enzymes. After two sequential screenings, 5 clones out of 5 times 10^4 exhibiting positive hybridization signals were isolated; one clone, named pDR7, had a 2.3-kb region encompassing the complete gene sequence. The hybridization of the whole coding sequence as a probe to the bacterial chromosomal DNA produced results consistent with the restriction maps of the isolated clone, thus confirming that the recombinant clone suffered no rearrangements during the cloning procedures.


Figure 2: Restriction endonuclease map of the genomic fragment in pDR7 plasmid carrying the gene coding for the P. furiosus protein. The arrows indicate the sequencing strategy, which employed synthetic oligonucleotides (PfN, PfC, Pf1, Pf2, Pf3, and Pf4) and universal M13 primers on fragments suitably subcloned (see ``Experimental Procedures'' for details).



Fig. 3shows the nucleotide sequence of the gene and the deduced primary structure of the P. furiosus protein. A 675-bp open reading frame (ORF) starting at ATG (position 106) and ending at TAA (position 783) as well as extended flanking regions were found. Upstream to the initiation codon, the putative archaebacterial TATA box (at positions 76-81) and the ribosome binding site (at positions 98-102) could be identified (Reiter et al., 1988). The 226 amino acid residues derived from the ORF matched up to the calculated size of the native protein whose N-terminal sequence lacks the initial methionine. The residues from Phe-141 to Cys-149 identify the FXXXXCXXC motif, which is a part of the active site of the enzymes that catalyze thiol/disulfide interchange reactions; the sequence CPYC from Cys-146 to Cys-149 is the canonical active disulfide of glutaredoxins. The sequence CQYC from residue 35 to residue 38 is not conserved in the active site of any of the known protein disulfide oxidoreductases.


Figure 3: Nucleotide sequence (numbering at right) of the gene coding for the P. furiosus protein and the deduced amino acid sequence (numbering at left). The underlined nucleotides represent putative archaebacterial TATA box (from 76 to 81) and ribosome binding site (from 98 to 102). The amino acid residues that identify the conserved active site of glutaredoxin (from 146 to 149) are in boldface.



When the primary structure of the P. furiosus protein was compared with GenBank using the Fasta program, similarity with the active site region of some glutaredoxins was detected. We aligned the sequence CPYC of the P. furiosus protein with the conserved active site sequences of glutaredoxins from different sources and maximized similarity by introducing gaps. The moiety of the P. furiosus protein from residue 122 was compared with the full primary structures of glutaredoxins (Fig. 4). Five residues, valine 140, cysteines 146 and 149 of the active site, proline 194, and glycine 202, are common among all nine proteins, whereas proline 147, aspartic 186, valine 193 and 197, and glycine 210 are common among all but one of the proteins (numbering refers to the complete sequence of the P. furiosus protein). The highest identity (40%) was with the protein isolated from the methanogenic archaeon Methanobacterium thermoautotrophicum; the lowest (23%) was with T4 glutaredoxin. Prediction of secondary structures of the P. furiosus protein, obtained by Chou-Fasman analysis, revealed that the sequence CPYC, located in a turn, is preceded by a beta-sheet (residues 137-144) and followed by a large alpha-helix (residues 151-168). Similar structural features have been identified in the active site region of all glutaredoxins isolated (McFarlan et al.(1992), and references therein).


Figure 4: Comparison of the amino acid sequences of different glutaredoxins. The alignment was performed as described under ``Results.'' The sequences were from the following sources (in parentheses): M. thermoautotrophicum glutaredoxin (McFarlan et al., 1992), E. coli glutaredoxin (Hoog et al., 1983), yeast glutaredoxin (Gan et al., 1990), bovine glutaredoxin (Papayannopoulos et al., 1989), rabbit glutaredoxin (Hopper et al., 1989), pig glutaredoxin (Gan and Wells, 1987), rice glutaredoxin (Minakuchi et al., 1994), and T4 glutaredoxin (Sjoberg and Holmgren, 1972). Positional numbers refer to the sequence of the P. furiosus protein. The residues identical to the sequence of the P. furiosus protein are in boldface. The boxed residues indicate the conserved active site sequence of glutaredoxin.



Insulin Reductase Activity and Thioltransferase Activity of the P. furiosus Protein

Fig. 5shows the reduction of insulin disulfides at 30 °C in the presence of increasing concentrations of pure P. furiosus protein and in its absence (the spontaneous precipitation reaction), DTT being the reducing agent that recycles the oxidized protein. The progress curve obtained for 5 µM protein (not shown) was identical to that for 1.2 µM, indicating that the assay was saturated.


Figure 5: DTT-dependent insulin reduction. The DTT-dependent reduction of bovine insulin disulfides was carried out as described under ``Experimental Procedures'' in the absence (circle) or in the presence of different concentrations of pure P. furiosus protein: bullet, 0.2 µM; , 0.6 µM; , 1.2 µM.



Glutaredoxin has an inherent GSH-dependent disulfide reductase activity (named the thioltransferase activity) that can be assayed by coupling to saturating concentrations of glutathione reductase, which reduces GSSG in the presence of NADPH (Axelsson et al., 1978). By using L-cystine as disulfide substrate, we demonstrated that the P. furiosus protein exhibits thioltransferase activity. The variations in activity with GSH and L-cystine concentrations showed that the maximal rate was obtained at 10 mM GSH and 2.5 mML-cystine; inhibition of the reaction rate was detected by further increasing the concentrations of the reactants. The thioltransferase activity of the P. furiosus protein showed a linear dependence on the protein amount up to 0.12 µM, inhibition being detected at higher protein concentrations (Fig. 6A); when the activity was assayed over the pH range of 6-9, a sharp optimum was found at pH 8.0 (Fig. 6B). Most glutaredoxins have been reported in the literature to have basic pH optima.


Figure 6: A, dependence of the thioltransferase activity on the concentration of pure P. furiosus protein. The standard assay mixture (see ``Experimental Procedures'') contained different amounts of protein. B, dependence of the thioltransferase activity on pH. The activity was assayed in sodium phosphate buffer (bullet) and Tris-HCl buffer () at the indicated pH values, as described under ``Experimental Procedures.'' In A and B, the activity is expressed as the net enzymatic velocity; each value is the average of two separate experiments.



High Level Expression of the P. furiosus Protein in E. coli

The expression vector pGlx was constructed by inserting the correct coding sequence obtained by gene amplification into the plasmid pTRC99A between the NcoI and XbaI sites, in frame with the initiation codon provided by the plasmid. In pTRC99A plasmid, transcription of recombinant genes is controlled by the strong hybrid promoter ptrc, derived from the fusion of the lac and trp promoters, and is still inducible by IPTG.

E. coli Rb791-competent cells were transformed with the newly constructed pGlx expression vector and allowed to grow in aliquots up to 0.5, 1, 1.5, 2, and 2.5 A cell densities; the expression of the P. furiosus protein was induced by the addition of 1 mM IPTG to the culture media, the induction time being 16 h in each case. The five extracts and the extract from E. coli cells transformed with the expression vector lacking any inserted sequence (the negative control) were assayed for the thioltransferase activity using the same protein amount: maximum activity was found in the extract from transformed cells grown at 1 A cell density. To further optimize the expression of the protein, transformed cells grown at 1 A cell density were exposed to 1 mM IPTG for increasing time lengths, and the thioltransferase activity was assayed on each extract as described; the results showed that the maximum level of expression was reached after an 18-h induction.

For preparative purposes, the crude extract from transformed cells grown at 1 A cell density and induced for 18 h with IPTG underwent two successive thermal precipitation steps (at 65 and 70 °C); most of the E. coli proteins became insoluble and were removed by centrifugation, whereas recombinant protein was recovered soluble and active in the supernatant. Following the thermoprecipitation steps, about 70-80% of the E. coli proteins were removed, yielding an almost homogeneous 24.8-kDa protein, as judged by SDS-PAGE (Fig. 7); minor contaminants were removed by a gel filtration chromatography. The recombinant protein and the native one were indistinguishable in pI, thermal stability, and catalytic activities.


Figure 7: SDS-PAGE analysis of the steps during the purification of expressed P. furiosus protein. Lane 1, E. coli crude extract; lane 2, sample after heat treatments; lane 3, sample from Superdex 75 column; lane 4, molecular mass standards. Samples (6 µg/lane), treated and run as described under ``Experimental Procedures,'' were stained with Coomassie Blue.




DISCUSSION

In the framework of a project aimed at studying protein disulfide oxidoreductases from Archaea, we purified a heat-stable redox protein of 24.8 kDa and pI 4.9 from the crude extract of the hyperthermophile P. furiosus. The purification conditions described yielded 0.5 mg of pure protein from 10 g of cell paste; this yield did not vary significantly when the purification was carried out in the presence of DTT. The low cellular amount of a protein poses a limit to its structural and functional characterization; therefore, we assessed a high level expression system of the recombinant P. furiosus protein cloned in E. coli by optimization of both the growth time of the transformed cells and the induction time with IPTG. Based on the consideration that a heat-stable protein expressed in a mesophilic host differs from the endogenous proteins for its resistance against heating, the extract of E. coli was enriched for the P. furiosus protein by two simple thermal precipitation steps. The expression system and the purification procedure described allowed us to obtain fully active protein in a very large amount: 18 mg of pure protein starting from 85 mg of E. coli crude extract. The crystallization of the P. furiosus protein is currently being carried out with the purpose of obtaining information about its three-dimensional structure.

The protein from P. furiosus described here is clearly a member of the family of protein disulfide oxidoreductases; the presence of the sequence CPYC and the demonstration of its thioltransferase activity prompt us to call it ``glutaredoxin-like'' protein. However, it is an unusual protein. All glutaredoxins described so far have the conserved active sequence located at their N terminus; the sequence CPYC is situated at the C-terminal portion of the P. furiosus protein, which shows sequence similarity with glutaredoxins from different sources (Fig. 4). The dithiol in the sequence CQYC at the N terminus of the P. furiosus protein (residues 35-38) is not conserved in any of the protein disulfide oxidoreductases described so far. We exclude the possibility that this dithiol could be considered a homologue to the additional dithiol that is present in all mammalian glutaredoxins with the conserved sequence CIGGC and whose function is not clear. Even if the canonical sequence CPYC is the best candidate for being responsible for the catalytic activity of the P. furiosus protein, we cannot rule out a contribution of the additional dithiol. The expression system described is highly suitable for site-directed mutagenesis studies that can provide precise information about which residues contribute to the catalytic activity and to what extent.

The molecular mass of the P. furiosus protein, 24.8 kDa, is unusual for most glutaredoxins, which have a typical size of 10-12 kDa. E. coli contains two glutaredoxins of 10 kDa and one glutaredoxin of 27 kDa, named Grx2, whose N-terminal sequence shows the conserved site CPYC (Aslund et al., 1994). One cannot simply assume that the P. furiosus protein described here is the homologue of Grx2; nevertheless, a class of large glutaredoxins may exist. The protein of 24 kDa isolated from the aerobic hyperthermophilic archaeon Sulfolobus solfataricus (Guagliardi et al., 1994), called thioredoxin, is likely to be the homologue of the P. furiosus protein; the two proteins have similar amino acid compositions and pI as well as comparable insulin reductase activity and thioltransferase activity. (^2)The chloroplasts of the unicellular green algae Scenedesmus obliquus contain a thioredoxin of 28 kDa (Langlotz et al., 1986; Langlotz and Follmann, 1987); the cyanobacterium Anabaena sp.7119 has a thioredoxin of 25.5 kDa, which shares amino acid content with the S. obliquus protein (Whittaker and Gleason, 1984). These proteins have been classified as thioredoxins based on their capability to stimulate several disulfide-containing enzymes. However, the S. obliquus protein does not show immunoreactivity with the E. coli thioredoxin, and fluorescence studies suggest that the tryptophan residues in the Anabaena protein are not in the vicinity of the active site, as typical of such residues in thioredoxins. The determination of the primary structures of the large redox proteins will be of great interest, as will the eventual elucidation of their functional specialization.

In Bacteria and Eucarya, protein disulfide oxidoreductases are involved in many fundamental cellular processes; protein disulfide isomerase is responsible for the catalytic rearrangements necessary for the correct folding of nascent protein molecules, whereas the thioredoxin system (thioredoxin, NADPH, and thioredoxin reductase) and the glutaredoxin system (glutaredoxin, GSH, NADPH, and glutathione reductase) have been proposed to participate in several different cellular processes through disulfide reductase activity. The information available on protein disulfide oxidoreductases from Archaea is too scanty to define their physiological function(s). The protein described here from P. furiosus and that from S. solfataricus can catalyze the reduction of disulfide bonds in proteins and small compounds; both proteins failed to reactivate scrambled RNase A and to form disulfide bonds in reduced denatured RNase A (data not shown). The protein isolated from the strictly anaerobic methanogen M. thermoautotrophicum serves as a hydrogen donor for the E. coli ribonucleotide reductase and catalyzes the DTT-dependent reduction of insulin disulfides but does not show thioltransferase activity (Schlicht et al., 1985; McFarlan et al. 1992).

The reducing system utilized by the archaeal protein disulfide oxidoreductases is also puzzling. We were unable to detect NADPH-dependent glutathione reductase in crude extracts of P. furiosus and S. solfataricus. To the best of our knowledge, the extracts of different Archaea are devoid of GSH or related peptides, even if the occurrence of other monothiols cannot be ruled out. McFarlan et al.(1992) suggested that the glutaredoxin from M. thermoautotrophicum may utilize a reducing system based on hydrogenase and ferredoxin. Ferredoxins and hydrogenases have been isolated from different Archaea (Adams(1993, 1994), and references therein), and in our opinion, the possibility exists that a ferredoxin-dependent reducing system might work in Archaea. Of course, this hypothesis must await further experimental support.


FOOTNOTES

*
This work has been carried out under a research contract with Farmitalia Carlo Erba S.r.l., Milano, Italy, within the Programma Nazionale di Ricerca sulle Tecnologie per la Bioelettronica del Ministero dell'Università e della Ricerca Scientifica e Tecnologica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z38068[GenBank].

§
To whom correspondence should be addressed: Dipartimento di Chimica Organica e Biologica, Università di Napoli, Via Mezzocannone 16, 80134 Napoli, Italy. Tel.: 39-81-704-1236; Fax: 39-81-552-1217.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; FPLC, fast protein liquid chromatography; HPLC, high pressure liquid chromatography; IPTG, isopropyl-1-thio-beta-D-galactopyranoside.

(^2)
A. Guagliardi, D. de Pascale, R. Cannio, V. Nobile, S. Bartolucci, and M. Rossi, unpublished data.


ACKNOWLEDGEMENTS

Prof. Mario De Rosa (Istituto di Biochimica delle Macromolecole, Università of Napoli) is acknowledged for providing the bacterial paste of P. furiosus.


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