©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Overproduction and Physical Characterization of SoxR, a 2Fe-2S Protein That Governs an Oxidative Response Regulon in Escherichia coli(*)

Jie Wu (1), William R. Dunham (2), Bernard Weiss (1)(§)

From the (1) Department of Pathology, the University of Michigan Medical School, Ann Arbor, Michigan 48109-0602 and the (2) Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109-1055

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

SoxR protein governs the soxRS (superoxide response) regulon of Escherichia coli by becoming a transcriptional activator when the cells are exposed to compounds that mediate univalent redox reactions, many of which produce superoxide as a by-product. SoxR was overproduced and purified to near homogeneity from a strain bearing an expression vector. It could bind specifically to the soxS operator even in the absence of RNA polymerase. The aerobically purified protein, which is readily autooxidized, could activate the transcription of soxS DNA even without exposure to known inducing agents. SoxR is a globular homodimer. It contains one [2Fe-2S] cluster per polypeptide chain, as demonstrated by optical and EPR spectroscopy combined with stoichiometric analysis of iron content, unpaired-electron-spin density, and reduction by dithionite. The protein is active in its oxidized ([2Fe-2S]) state. The presence of a prosthetic group capable of univalent redox reactions may help to explain the activation of the regulon in vivo by compounds that can mediate such reactions.


INTRODUCTION

The transcriptional regulators, SoxR and SoxS, govern a regulon of Escherichia coli that responds to superoxide generators such as paraquat (methyl viologen) (1, 2) . The soxRS regulon and the oxyR regulon (which protects against HO) are the two best studied bacterial regulatory systems for adaptive defenses against oxidants (3) . The soxRS regulon includes the following genes of known function: sodA, encoding Mn-superoxide dismutase; nfo, encoding endonuclease IV, a DNA repair enzyme; zwf, encoding glucose-6-phosphate dehydrogenase, which regenerates the reducing equivalents (in the form of NADPH) that are consumed during redox cycling of the inducers; micF, whose mRNA inhibits the porin gene, ompF; fpr, encoding ferredoxin (flavodoxin):NADPH oxidoreductase, which assists in the reduction of Fe-S proteins (4) ; and fumC, encoding an oxidatively stable fumarase (5) .

The induction of the soxRS regulon occurs in two steps (6, 7) . SoxR responds to an oxidative signal and activates the transcription of soxS. The overproduced SoxS then activates the transcription of the target genes of the regulon. This cascade increases sensitivity through signal amplification. Although SoxS is a member of the AraC family of transcriptional activators, it lacks a region corresponding to the sensor domains of its relatives (8, 9) . The following findings suggested that SoxR provides this sensor function. The action of SoxR precedes that of SoxS, some soxR mutants have a regulon-constitutive phenotype, and SoxR has a cluster of four cysteines that may be part of a redox-sensing center (6, 7, 8, 9) .

In preliminary experiments (10) , we obtained active SoxR with an average of 1.0 iron atom per chain and the optical spectrum of an iron-sulfur protein. A recent report (11) described the purification of SoxR that had 1.6 iron atoms per polypeptide and a similar spectrum. The low iron content was consistent with the well known instability of many Fe-S clusters. The number of iron atoms per Fe-S center and the number of clusters per polypeptide were not determined. In this study, we report the large scale production and purification of SoxR that contains a full complement of Fe-S clusters, a prerequisite for crystallographic and other physical studies. We determine some of its molecular parameters, and we show that the active protein is a globular homodimer containing one [2Fe-2S] center per polypeptide chain. Thus, SoxR is related to a class of well characterized electron transport proteins, the [2Fe-2S] ferredoxins. This identification of the redox center provides an important insight into the mechanism of activation of the soxRS regulon by oxidative stress.


EXPERIMENTAL PROCEDURES

Strains and Plasmids

E. coli strain BL21(DE3) and plasmid pET-11 (12) were obtained from Novagen. Plasmid pHE6:: soxR, a derivative of pHE6 (13) , will be separately described.

Molecular Biological Methods

General methods for cloning and for electrophoretic analysis of polynucleotides and proteins were as described (14, 15) . DNA concentrations were estimated by staining with ethidium bromide (14) or by fluorimetry with Hoechst 33258 dye (16) .

Overproduction of SoxR

High density bacterial growth was performed as described (17) , with the following modifications. A 60-ml saturated culture of E. coli BL21(pET11K- soxR, DE3) was inoculated into 3 liters of medium containing kanamycin (50 µg/ml) and incubated at 37 °C under oxygen. At an ODof 21 (11 h), the culture was rapidly cooled to 15 °C, and 2 ml of a 20% IPTG() solution were added, followed 6 h later by 600 mg of rifampicin. The cells were harvested 23 h after induction (OD= 32, pH 6.6). The cell paste (130 g) was washed and resuspended in an equal volume of 10% sucrose, 10 mM TrisHCl buffer (pH 8.0), dripped into liquid N, and stored at 80 °C.

Overproduction was also achieved in 500-ml flasks on a water bath shaker in room atmosphere. One ml of a saturated culture was added to 100 ml of medium in each flask. The temperature shift was performed at a cell density of 5 10ml. Rifampicin was omitted. The yield was 1.7 g of cell paste per flask.

Purification of SoxR

All operations were performed at 0 to 4 °C in a standard buffer containing 20 mM MOPS/KOH (pH 7.6), 1 mM dithiothreitol, and 10% glycerol. Precipitates were collected by centrifugation at 27,000 g for 10 min after 20 min of stirring. Twenty grams of the frozen cell suspension were sonicated in 40 ml of the standard buffer containing 0.2 M KCl and 0.0025% phenylmethylsulfonyl fluoride, and cell debris was removed by centrifugation at 33,000 g for 15 min. NaCl (5 M) and polyethyleneimine-HCl (5%, pH 7.0) were added to final concentrations of 0.1 M and 0.225%, respectively, and the precipitate was removed by centrifugation. An equal volume of a saturated solution of (NH)SOwas slowly added. The precipitate was dissolved in 40 ml of standard buffer, precipitated again with 60 ml of a saturated (NH)SOsolution, and redissolved in 200 ml of standard buffer containing 0.2 M KCl (loading buffer).

Whatman P-11 phosphocellulose was equilibrated with the loading buffer and packed into a column (7.6 cm 1 cm) to which the protein solution was applied followed by 12 ml of the loading buffer. Elution was performed with 60 ml of the loading buffer containing a linear gradient of from 0.2 to 1.0 M KCl. SoxR was eluted in a single peak at 0.5 to 0.6 M KCl. It was detected by its reddish brown color and electrophoretic mobility.

DNA Substrates

The soxS promoter segment used for electrophoretic mobility shift assays was an EcoRI- BstYI fragment of an M13mp19:: soxRS plasmid that had a terminal deletion of most of the soxR gene (8) ; it contained nucleotides 459-586 of the published sequence (8) . The control ( soxR) DNA was an EcoRI- SmaI fragment of a similar plasmid that had an intact soxR gene; it contained nucleotides 977-1099. The soxS promoter DNA used for transcription assays extended from nucleotides 421-594. It was generated by a polymerase chain reaction (15) that employed a fragment of plasmid pUC18:: soxS (6) as a template and the following oligonucleotides as primers: TAAGCGGCTGGTCAATATGC and GCGGGGTAATTTCTTTTCCA.

Electrophoretic Band Shift Assays

Mobility shift DNA binding assays using low ionic strength PAGE were performed as described elsewhere (15) . Prior to electrophoresis, the P-labeled DNA fragments (7500 cpm each) were incubated with from 0 to 200 ng of SoxR protein for 20 min at 30 °C in 15 µl of a solution containing 133 µg/ml poly(dI-dC), 300 µg/ml bovine serum albumin, 12% glycerol, 10 mM KCl, 1 mM dithiothreitol, and 12 mM HEPES/NaOH buffer (pH 7.9).

Transcription Assays

SoxR was diluted in a solution containing 20 mM TrisHCl buffer (pH 8.0), 100 mM KCl, 1 mM dithiothreitol, and 50 µg/ml bovine serum albumin. SoxR (5-50 ng in 3 µl of diluent) was added to 18 µl of a solution containing 15 nmol of DNA template and 0.16 mM each of ATP, GTP, and CTP, 40 mM TrisHCl buffer (pH 8.0), 10 mM MgCl, 50 mM KCl, 1 mM dithiothreitol, and 1 mg/ml bovine serum albumin. After the mixture was incubated for 10 min at 37 °C, 0.15 unit of E. coli RNA polymerase-holoenzyme (Epicentre Technologies) was added in 3 µl. After an additional 10 min at 37 °C, 3 µl of heparin sulfate (1 mg/ml) and 3 µl of 80 µM -[P]UTP (3-30 Bq/mmol) were added simultaneously. The mixtures were incubated at 37 °C for 15 min and treated with phenol. The RNA products were precipitated with ethanol and analyzed by electrophoresis in an 8% polyacrylamide, 7.7 M urea gel (15) . Radioactivity was measured with the Ambis Corp. (San Diego, CA) radioanalytic imaging system. One standard unit of activity is defined as the incorporation of 1 fmol of UTP under these conditions. The activity was proportional (±5%) to the concentration of SoxR in the stated range.

Protein Analyses

Protein concentrations were measured with the Pierce Coomassie Blue dye reagent, using bovine serum albumin as a standard (18) . Values for SoxR were multiplied by 0.323 to agree with those obtained with a Beckman 7300 Amino Acid Analyzer at the W. M. Keck Foundation, Yale University. N-terminal sequences were determined by Edman degradation, using an Applied Biosystems model 470 sequencer at the University of Michigan Protein and Carbohydrate Structure Facility.

Gel Permeation Chromatography

A column (1-cm diameter, 35-ml volume) of Bio-Gel P-60 (Bio-Rad) was equilibrated and operated at 4 °C with a solution containing 0.25 M KCl, 10 mM dithiothreitol, 20 mM MOPS/NaOH buffer (pH 7.6), and 10% glycerol. SoxR (45 µg) or other proteins (1 mg each) were applied in a volume of 0.1 ml. The void volume was determined with bovine catalase (240 kDa). Of the material applied, 37% of the SoxR protein and 24% of its original activity were recovered in the combined peak fractions. The markers used were ovalbumin, pancreatic DNase, bovine chymotrypsinogen, and horse heart cytochrome c.

Sedimentation Analysis

Band sedimentation was performed in 4.5-ml solutions containing 0.25 M KCl, 10 mM dithiothreitol, 20 mM MOPS/NaOH buffer (pH 7.6), and a linear gradient of from 10 to 30% glycerol. The protein samples were 0.3 to 0.5 mg. Ovalbumin and chymotrypsinogen were the standards. Centrifugation was in a Beckman SW50.1 rotor at 48,000 rpm for 26 h at 4 °C.

Physical Parameters

Methods of calculation for the Stokes radius, sedimentation coefficient, frictional ratio, and Mof native SoxR were as in Weiss (19) . A value of 0.736 was assumed for the partial specific volume of SoxR (20) .

Metal Analyses

Precautions were taken to reduce contaminating metal ions (21) . Metal analyses were performed by inductively coupled plasma atomic emission spectrometry in the Department of Geological Sciences at the University of Michigan. An ultrafiltrate served as a blank sample; it contained <10% as much iron as the SoxR solution.

Reductive Titrations

Titrations of SoxR with sodium dithionite (22, 23) were performed at 4 °C under argon. Residual oxygen was removed with protocatechuic acid and protocatechuate dioxygenase (a generous gift of D. Ballou) (24) , which were added to 100 µM and 1 µM, respectively. The dithionite solution was standardized with FAD.

EPR Spectra

Methods used for EPR spectroscopy (25) and computer programs for data reduction (26, 27) were as described previously.


RESULTS

Cloning of soxR in an Expression Vector

A promoterless DNA fragment containing the ribosome binding site and complete coding sequence of soxR was inserted into plasmid pET-11, thereby placing the soxR gene downstream from a phage T7 promoter under lac operator control (Fig. 1). The plasmid was unstable in high density cultures in which ampicillin, which is readily destroyed by -lactamase, was used as the selective agent (12) . Therefore, a kanamycin resistance gene was inserted. The resulting strain was >99% stable as determined by its ability to be killed by IPTG (12) .


Figure 1: Structure of pET11K- soxR, a plasmid used for the overexpression of SoxR. The open bars are DNA segments that were cloned into the expression vector pET-11. One contains the soxR gene on a 620-bp BamHI fragment of plasmid pHE6:: soxR, which was inserted into a BamHI site. The other contains the kanamycin resistance gene of plasmid pUC4K (28) on a 1.3-kb EcoRI fragment that was cloned into the PstI site within the bla gene. Tis a phage transcriptional terminator. The sequence shows the fusion of the T7 gene 10 promoter/ lac operator region (P) of the vector to the cloned segment containing the soxR ribosomal binding site ( RBS) and structural gene. The DNA to the left of the BamHI site is from pET-11. The sequence in brackets is a BamHI- SmaI fragment from pHE6 (13). Downstream of the soxR gene are 61 additional base pairs of contiguous chromosomal DNA connected by (dG)to an 87-bp EcoRI- PvuII segment of M13mp18 DNA (8).



Activity in Vivo of the Cloned soxR Gene

To ensure that our cloned soxR gene had not mutated, we tested for its ability to restore to a soxR9::cat mutant (6) the capacity of its zwf (glucose-6-phosphate dehydrogenase) gene to be induced by paraquat, a function that is mediated by the soxRS regulon (1, 2) . The plasmid enabled a 10-fold induction of the enzyme, comparable to that seen in wild type cells.

Overproduction of SoxR

The use of a vector with an IPTG-inducible rather than a thermoinducible promoter, enabled the overexpression of SoxR at low temperature, which favors the synthesis of correctly folded, soluble, overexpressed proteins (29) . At 30 or 37 °C, >50% of the SoxR produced from plasmid pET11K- soxR was insoluble; however, at 15 °C, almost all of the SoxR protein produced could be recovered in a soluble fraction (Fig. 2).


Figure 2: Overproduction ( A) and purification ( B) of SoxR as monitored by SDS-PAGE. A, a fermentor culture of strain BL21 (pET11K- soxR, DE3) was sampled at 0, 18, and 23 h after treatment with IPTG at 15 °C. Samples of equal cell mass were applied to the gel after lysis and denaturation. B, lane I, sonicate supernatant; II, polyethyleneimine supernatant; III, ammonium sulfate precipitate; IV, phosphocellulose eluate. Lanes I to III each contained proteins derived from 1 µl of sonicate. Lane IV contained 10 µg of SoxR. Markers were chymotrypsinogen (25 kDa) and lysozyme (14 kDa).



Purification

Only one chromatography step was needed to purify SoxR (Fig. 2 B). Its high pI enabled it bind to phosphocellulose at a high salt concentration required for its stability. The yield was 1.5 mg of purified SoxR per g of wet cells. Its specific activity was 10 units/ng of protein (transcriptional activation assay).

The first five N-terminal amino acids of the purified protein matched those predicted by the DNA sequence of soxR (8) , indicating that there was no amino-terminal processing. A secondary band detected by SDS-PAGE (Fig. 2 B, IV), had the mobility and N-terminal sequence expected of undissociated SoxR dimer.

General Properties

Purified SoxR containing 1 mM dithiothreitol was stable only for about 1 week at 4 °C. Preparations containing 10 mM dithiothreitol were stable for about 1 month at 4 °C under argon or for at least 2 months at 20 °C in 50% glycerol. At salt concentrations <0.2 M or at temperatures >10 °C, the reddish brown solution formed an insoluble white precipitate within a few minutes. Fast-frozen samples that were stored at 80 °C lost 15% of their activity on thawing. Although SoxR was reported to have a solubility 200 µg/ml (11) , under our conditions it was soluble at >10 mg/ml.

DNA Binding Activity

Purified SoxR bound specifically to a fragment of soxS DNA extending 62 base pairs upstream of the transcriptional start site. The electrophoretic mobility of the DNA was retarded 11%, even in the absence of RNA polymerase. Under the conditions given (see ``Experimental Procedures''), the binding constant of SoxR dimer to soxS DNA (determined as the concentration of SoxR required for retardation of 50% of the DNA) was between 0.4 and 2.0 10 M. The results (not shown) were similar to those obtained by others (11) and confirmed the activity of our preparation.

Transcriptional Enhancement

Purified SoxR was tested for its ability to activate the transcription of the soxS gene. The 173-bp linear soxS DNA template started 67 nucleotides upstream of the transcriptional start site. The one-cycle transcription assay employed heparin to block reinitiation during the elongation phase, thereby increasing the relative yield of full length transcripts. The results (Fig. 3) indicated that transcription from the SoxS promoter was dependent on the addition of SoxR. If SoxR were added after heparin, there was no transcriptional enhancement (results not shown). Therefore, SoxR functions in transcription initiation.


Figure 3: Activation of soxS transcription by purified SoxR. A one-cycle transcription assay was performed with a soxS DNA template and increasing amounts of SoxR protein (see ``Experimental Procedures'').



In vivo, SoxR requires activation by treatment of cells with oxidants. However, purified SoxR was active in vitro without further treatment (Fig. 3) (11) . We were unable to increase the activity of our preparation by incubating it with potassium superoxide or with superoxide generating systems (phenazine methosulfate plus NADPH and O, or 6-hydroxydopamine plus O), even in the presence of catalase to remove any HOthat might inactivate the protein (results not shown).

SoxR Is a Homodimer

Transcriptional activity and protein comigrated in a single peak during gel permeation chromatography. Although the molecular mass of the polypeptide is 17 kDa (8) , the native protein had an elution volume between that of pancreatic DNase I (31 kDa) and ovalbumin (43 kDa), and its sedimentation rate was between that of ovalbumin and chymotrypsinogen (25 kDa). The physical parameters of the native protein are as follows: Stokes radius, 2.81 nm; diffusion coefficient ( D), 7.65 10cms; sedimentation coefficient ( s), 2.71 S; frictional ratio ( f/f), 1.26; M(calculated from D and s), 32,700. Thus, SoxR is a globular homodimer in its active form.

Metal Content

Our SoxR preparation contained 2.0 atoms of iron per polypeptide chain. Values for other metals were as follows: zinc, a ubiquitous contaminant (21) , 0.39; copper, 0.34; and cobalt, molybdenum, and magnesium, 0.10.

Optical Spectrum

At peak wavelengths, the extinction coefficients (mMcm) for the SoxR polypeptide were as follows: = 53.5, = 24.5, = 12.7, = 12.4, and (shoulder) = 8.0. The spectrum was characteristic of a [2Fe-2S] protein (30) . The ratio of the absorbance of other peaks to that at 276 nm is higher than that previously observed (11) , reflecting its higher iron content.

Reductive Titration with Dithionite

Reduction by dithionite (Fig. 4) produced a decrease in absorbance above 300 nm as observed with [2Fe-2S] ferredoxins (23) . When the reduced protein was aerated, the original spectrum was almost completely restored within 2 min (Fig. 4, curve d), indicating that SoxR is readily autooxidized and that the spectral changes had been caused by the reversible reduction of the protein and not by its denaturation.


Figure 4: Reductive titration of SoxR by sodium dithionite. A solution of 0.28 mM sodium dithionite in 20 mM MOPS buffer (pH 7.6) was added incrementally to SoxR (0.37 mg/ml) under anaerobic conditions in a 1-cm path length cuvette. Spectra were recorded: a, before treatment; b, after partial titration; c, after full titration; and d, after re-exposure to air for 2 min. Inset, Titration of a SoxR solution containing 34 µM bound Fe.



Oxidized [2Fe-2S] and [4Fe-4S] centers each accept one electron from dithionite (23) . When SoxR protein was titrated anaerobically with dithionite, 0.5 reducing equivalent was accepted per gram atom of iron (Fig. 4, inset). These results strongly suggest that each Fe-S center contains 2Fe. Because there are only 2Fe and 4 cysteines per chain of SoxR, there should be one [2Fe-2S] center per chain.

EPR Spectrum

Untreated SoxR produced no EPR signal, thereby ruling out the presence of a [3Fe-3S] or [3Fe-4S] cluster (31) . A sample reduced by dithionite gave EPR signals with g values of 2.01, 1.92, and 1.90 (Fig. 5), which are consistent with those of an S = 1/2 [2Fe-2S] center and close to those produced by the [2Fe-2S] cluster in E. coli fumarate reductase (2.03, 1.93, 1.92) (32) . The almost axial spectrum resembles that first described for the hydroxylase type of [2Fe-2S] protein, which is exemplified by putidaredoxin and adrenodoxin ( g = 2.02, 1.94, 1.94) (33) .


Figure 5: EPR spectrum (25 K) of SoxR after 50% reduction by dithionite. The data ( solid line) are superimposed by a simulation ( dashed line) with g values: 1.903, 1.922, 2.007, and line widths given in g units: 0.0086, 0.0065, and 0.0041, respectively. The incident microwave power was 0.4 milliwatt; the field modulation amplitude was 0.5 mT.



EPR spectroscopy was performed on samples of SoxR that had been titrated spectrophometrically with dithionite. For a fully titrated solution of SoxR containing 45 µm of bound iron, the density of unpaired electron spins ( i.e. the concentration of Fe-S centers) was 22 µM; for a half-titrated one, it was 11 µM. These results confirmed that there are 2Fe per Fe-S cluster.

Sequence Similarities

Using Genetics Computer Group software (34) , we scanned the SWISS-PROT data base (35) for Fe-S proteins with sequences similar to a 20-amino acid segment of SoxR encompassing the cysteine cluster. The greatest similarity was found with the [4Fe-4S] centers in bacterial ferredoxins, almost all of which share the following consensus sequence with SoxR around the first two cysteines: D XC(I,L,V) XCG. However, the spacing of the cysteines in SoxR (C XC XC XC) is unique; the bacterial ferredoxins, e.g. have two amino acids separating the second and third cysteines. MerR, a Hg-binding protein to which SoxR seems most closely related (9) , has little homology to SoxR in this region, has only three cysteines (C XC XC), and does not contain an Fe-S cluster.


DISCUSSION

Active SoxR is a homodimer. Because each chain contains a DNA-binding (helix-turn-helix) motif (8, 9) , the protein is likely to recognize a repeated sequence in DNA. Indeed, the soxS operator, which covers most of the promoter region (11) , contains an 18-nucleotide palindrome (8) . The Fe-S clusters in SoxR are probably coordinated to all four of its cysteines, as is the case for most [2Fe-2S] proteins of known structure. However, there are exceptions (31, 36) , and the unusual spacing of the cysteines in SoxR adds to our uncertainty. It is also possible that Fe-S clusters might bridge the two polypeptide chains.

The superoxide response ( soxRS) regulon was so named because it is induced by superoxide generators like paraquat. It was therefore unexpected that SoxR, when purified from cells that were not exposed to these agents, was already active (11) (this work) and that this activity was not enhanced in vitro by superoxide generators. However, recent evidence (reviewed in Liochev and Fridovich (4) ) suggested that SoxR is likely to be activated in vivo through an oxidative chain rather than by superoxide directly.

Although the regulon is induced by univalent redox compounds, it is not induced by HO(37) , which primarily undergoes two-electron redox reactions in vivo. It is therefore of great significance that we found SoxR to contain [2Fe-2S] clusters, prosthetic groups that are capable of reversible univalent redox reactions. The most likely hypothesis is that SoxR is normally in a reduced ([2Fe-2S]) state in vivo and is activated by oxidation to the [2Fe-2S]state. This can occur upon exposure of the cell to superoxide generators and other univalent oxidants, or perhaps by any stress that decreases the availability of the NADPH and reduced flavodoxin or ferredoxins that may keep SoxR reduced (4) . In vitro, activation may occur rapidly by autooxidation. However, a crucial item of supporting evidence is lacking, namely, that the protein is inactive in its reduced form. So far, this demonstration has eluded us, as well as others (11) , because of the technical difficulty of performing multistep microscale assays under stringent anaerobic conditions.


FOOTNOTES

*
This work was supported by National Science Foundation Research Grant MCB-9304092. Computer services were provided by the General Clinical Research Center at the University of Michigan, funded by the National Center for Research Resources, National Institutes of Health Grant M01RR00042. 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.

§
To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, 1301 Catherine Rd., Ann Arbor, MI 48109-0602. Tel.: 313-764-2212; Fax: 313-763-6476.

The abbreviations used are: IPTG, isopropyl--D-thiogalactopyranoside; MOPS, 3-( N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We gratefully acknowledge the helpful advice of Dr. David P. Ballou.


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