The expression of a synthetic rainbow trout metallothionein gene in E. coli

178

Biochimica et Biophysica Acta, 1048 (1990) 178-186

Elsevier BBAEXP 92036

The expression of a synthetic rainbow trout metallothionein gene in E. coli Peter Kille

1 Paul Stephens 2, Anthony Cryer 1 and John Kay

1

J Department of Biochemisto, University of Wales College of Cardiff Cardiff and 2 Department of Gene Cloning, Celltech Ltd, Slough (U.K.)

(Received21 September1989)

Key words: Recombinantprotein; Metallothionein; Enzymepurification; E. coli expression; Cd stabilisation; Immunochemicalcharacterization;(Rainbow trout) A synthetic gene for rainbow trout metallothionein was constructed and inserted into a dual origin plasmid where expression was induced by a temperature shift in a proteinase-deficient strain of Escherichia coli. The recombinant protein was purified to homogeneity, and a partial amino acid sequence was determined to confirm its identity. Its immunochemical characteristics were similar to those of native metallothionein from rainbow trout. The amounts of recombinant metallothionein produced were quantified in soluble cell extracts by ELISA. Low concentrations were detected when growth was performed either in L-broth or defined (GMM-II) medium. Supplementation of the medium with zinc or copper had no effect on the amount of metaUothionein produced. By contrast, when cadmium was included in either L-broth or GMM-II medium, much higher concentrations of the protein within the cells (approx. 1 3 / ~ g / m g soluble cell protein) were detected. This stabilisation of the protein by metal reconstitution in vivo is considered in relation to the selective u p t a k e / e x c l u s i o n of metals by the cells and its significance for the scavenging of certain precious or toxic heavy metals is discussed.

Introduction

Metallothioneins (MT) are a family of cysteine-rich, low molecular weight, metal-binding proteins that are distributed widely in nature, in species ranging from single-cell organisms to humans [1]. They are generally considered to have an important role in the homeostasis of essential metal ions, e.g., zinc and copper [2], as well as in the detoxification of heavy metals such as cadmium and mercury. In addition to their cellular importance, the possibility of using metallothionein as a carrier of radioactive isotopes for therapeutic purposes has also been considered [3]. Thus, the nature of the interactions of particular metals with the protein is of considerable interest. In this regard, MT is an ideal protein for the examination of structure/activity relationships by sitedirected mutagenesis because of its small size, its abundance of cysteine residues and the paucity of aromatic residues, histidine and arginine. The efficient expression of yeast MT in homologous [4] and heterologous [5] systems has been reported previously. In E. coli, it has been shown that yeast MT

Correspondence: J. Kay, Department of Biochemistry, University of Wales Collegeof Cardiff, P.O. Box 903, Cardiff, CF1 1ST, U.K.

was a relatively stable product, with a half-life of approx. 15 min. By contrast, attempts to produce recombinant vertebrate metallothioneins [6,7] achieved only limited success. However, these proteins have little sequence homology to yeast MT, which may be reflected in a generally different conformation and stability in E. coli. Since previous work in this laboratory has been concerned with possible roles of MT in affording protection to certain species of fish against the toxic effects of cadmium [8,9], our attention has been focussed primarily on piscine metallothioneins. These do have considerable homology with, but yet are distinct from, the corresponding mammalian MTs [10-12]. In the present report, the construction of a synthetic gene for rainbow trout M T is described. The temperature-dependent expression of this gene within a dual origin vector [13], in E. coli is examined together with the isolation and characterisation of substantial quantities of the recombinant protein. Materials and Methods Bacterial strains and plasmids

Plasmid constructions were made in E. coli JM101 [14] and gene-expression studies were performed in E. coli 1B392 LonA1 (a lon deletion of 1B392, 15).

0167-4781/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)

179 Routinely, cultures were grown in Luria Broth (LB) containing Ampicillin (100 /~g/ml) and in GMM-II medium (40 mM Tris, 5.7 mM 2-glycerophosphate, 543 mM glycerol, 18.7 mM NH4C1, 5.7 mM K2SO4, 13.4 mM KC1, 0.07 mM CaC12, 0.2 mM MgC12 and Trace elements [16]) or LB for gene expression. The dual-origin expression vector pPW1 is a derivative of pMG 196 [17] in which the Shine-Dalgarno homology has been extended in order to increase expression levels from the trp promoter.

Synthetic gene synthesis DNA Oligonucleotides were synthezied on an Applied Biosystems synthesiser (Warrington, U.K.) and subsequently purified by HPLC. DNA manipulations and the production of plasmids were performed according to the methods of Maniatis et al. [18]. Restriction enzymes, ligases and kinases were obtained from BDH Chemicals (Poole, U.K.) whilst agarose gel reagents were obtained from Sigma Chemical Co. (Poole, U.K.). Plasmid sequencing was performed using the Sequenase Version 2.0 kit (United States Biochemical Corporation, Cleveland, OH, U.S.A.) following alkaline-denaturation of the double-stranded templates. 35S-labelled dATP was obtained from Amersham International (Amersham, U.K.) and other reagents were from Sigma Chemical Co. '(Poole, U.K.).

Competititve ELISA Samples for use in a competitive ELISA for the quantitative determination of MT were prepared as follows: Bacterial cells (1 ml cultures) were pelleted by centrifugation in a Microfuge (BDH Apparatus, Poole, U.K.) for 3 min, washed twice with phosphate-buffered saline (PBS) containing Tween (0.05% (w/v)) and resuspended in PBS/Tween. Samples were lysed by sonication (three 10 s pulses) using a cell disruptor Model B-30 at setting 3.5 (Branson Sonic Power Co., Danbury, CT, U.S.A.). Insoluble debris was removed by centrifugation in a Microfuge for 10 min. The concentration of MT in the supernatant samples prepared in this way was determined using a competitive ELISA, the details of which have been described previously [19]. Standard curves were prepared using either naturally occurring MT from Rainbow Trout (purified to homogeneity as described previously, [20]) as the soluble competitor or recombinant protein itself, once it became available. For the assays, metallothionein (between 15 and 22 ng) was coated onto each well overnight and standard curves were prepared by incubation of soluble competing antigen (102-107 pg) with antiserum to MT at a final dilution of 1/4000. The polyclonal antisera to homogeneous Rainbow Trout MT used were raised in mice as described previously [191. Protein concentration in the bacterial cell extracts was determined using a Bio-Rad assay kit (Bio-Rad,

Richmond, CA, U.S.A.). The manufacturer's micro-assay was adapted by mixing 160 #1 of sample (diluted appropriately) with 40 #1 of the dye reagent in wells of a 96-well microtitre plate (Dynatech Laboratories, Billingshurst, U.K.). Absorbance at 620 nm was measured using an Anthos 2001 plate reader (Anthos Labtec Instruments, Salzburg, Austria). Protein standards contained bovine serum albumin (10-40/,g/ml).

Purification of the recombinant protein Cultures of 1B392 LonZ~l (5 x 1 litre) hosting the pPWRt vector were incubated in LB at 30 ° C. At midlog phase, the cultures were immersed in a 68 °C water bath to raise the temperature to 42°C for plasmid induction. After a further incubation for 30 min at 37 ° C, cadmium was introduced into the medium at a final concentration of 300 #M. Incubation was then continued at 37 °C for a further 15 h at which time the cells were harvested by centrifugation at 3000 x g for 10 rain at 4 ° C. The cells were washed twice by resuspension in 500 ml of 10 mM sodium phosphate buffer (pH 7.4) containing 0.15 M KC1 with a final centrifugation at 5000 x g for 10 lnin at 4°C. The pelleted cells (13.6 g) were resuspended in the same buffer supplemented with 3 mM mercaptoethanol, and lysed by sonication (at setting 7) for 40 s / g wet weight of cells. Streptomycin (0.375 ml of a 10% (w/v) solution per g wet weight of cells) was added and the insoluble debris was removed by centrifugation at 18000 × g for 45 min at 4 ° C. The supernatant was then concentrated by ultrafiltration over a PM-10 membrane (Amicon, Stonehouse, U.K.) with retention of most of the Cd above the membrane. Subsequent purification of this concentrated cell supernatant by gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose was carried out as described previously [20].

Metal analysis, amino acid composition and N-terminal sequencing Cd, Zn and Cu were determined by atomic absorption spectrometry at 228.8, 213.9 and 324.8 nm, respectively, using a Varian model AA 275 fitted with background corrector (Varian Instruments, Walton-onThames, U.K.). Samples of protein for amino acid analysis were hydrolysed in 6 M HC1 in vacuo as described previously [20]. Recoveries of threonine and serine were calculated by extrapolation to zero time. Cysteine was determined as cysteic acid following oxidation of protein samples with performic acid [21]. SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli [221. Apo-metallothionein was prepared by removal of the metal ions (cadmium) from MT either by FPLC on a reverse-phase HR 5/5 column (Pharmacia, Milton Keynes, U.K.) eluted with acetonitrile containing 0.1%

180 trifluoroacetic acid (pH 1.8) or by exposure of the holo-protein to 3.5 M-guanidine/HC1 in 0.1 M HC1 [23] followed by chromatography on a column of Sephadex G-25. Blocking of the cysteine residues in the apo-protein by vinylpyridine was performed as described previously [24]. For amino acid sequence analysis, a sample of desalted pyridyl-ethylated protein was subjected to 24 cycles of Edman degradation in an Applied Biosystems Model 470A Protein Sequenator. The PTH-derivatives obtained were identified by HPLC using a methanol/ water gradient system as described previously [24]. Results

Construction of a synthetic M T gene The complete sequence of a naturally occurring metallothionein from Rainbow Trout has not been determined at the protein level although a partial (Nterminal) sequence has been reported [24]. The latter was in complete agreement with the sequence predicted from two full length cDNA clones isolated from a rainbow trout hepatoma cell line [10] that encoded two similar but distinct MTs. Proteins corresponding to these genes have not, however, been obtained or characterised. DNA oligonucleotides were therefore synthesized that

coded for both strands of the MT-B gene described by Bonham et al. [10] with the exception of the codon (GAC) for Asp in position 39 of the predicted protein which was replaced with a Gly (GGC). The latter is the (predominant) residue that is present in this position not only in the MT-A gene in the Rainbow Trout hepatoma cells [10] but also in MT from other species of fish [11,12]. The final construct with the organisation of the constitutive oligonucleotides after annealing is shown in Fig. 1. The ClaI/EcoRI gene monomer was extracted from a horizontal 2% low melting point agarose gel, ligated into expression vector pPW1 and the resultant mixture was used to transform JM101. Recombinant colonies were screened for insertion and orientation of the MT gene by restriction enzyme analysis. The sequence of the synthetic gene was confirmed by nucleotide sequencing using a plasmid based primer.

Expression of recombinant rMT Plasmids p M G 196 and pPW1 both contain two origins of replication (ori, Fig. 1), the low copy number ori pSC101 and associated par sequence, which ensures high plasmid stability in the absence of antibiotic selection and the high copy number ColEl-derived ori which is under the control of ?~pR and the c1857-coded temperature sensitive ~ repressor [15]. A temperature shift from 37 to 42 °C inactivates the X repressor and allows

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II Fig. 1. Structure of p P W R t plasmid. (a) Eight synthetic oligonucleotides were synthesised corresponding to both strands of the Rainbow Trout MT-B gene [10]. Oligonucleotides 1 - 4 spanned the entire length of the sense strand, while oligonucleotides 5 - 8 contained the antisense strand. After annealing, the monomer was isolated for ligation (see text). (b) The ClaI-EcoRl m o n o m e r was inserted by ligation with T 4 ligase, into pPW1 to generate a 7.1 kb plasmid (pPWRt).

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Fig. 2. (a) Growth of E. coli 1B392 LonA1 (transfected with pPWRt) in GMM-II medium in the absence (zx) and presence of cadmium at 100 (o) and 140/tM (~). In addition, concentrations of 20, 40, 60 and 80/tM cadmium produced identical results to those represented by the 100/~M Cd line. Similar responses were measured for E. coli transfected with the (control) pMG 196 plasmid (not shown). (b) Expression of recombinant rainbow trout metallothionein in E. coil grown in GMM-II medium. Induction was performed by heat stock at mid-log phase when E610 = 0.40. Cells were then maintained at 37 ° C in the absence (zx) or presence of 100 ~M cadmium (o), zinc (A) or copper (11), added exactly 30 rain after induction. Cultures were then analysed at the indicated times for MT in the soluble cell extracts by ELISA.

the ColEl-derived ori to become active and the copy number to increase approx. 30-fold [13]. Accordingly, pPWRt was transformed into the expression host 1B392 LonA1 maintained in GMM-II medium. After heat induction, the levels of recombinant MT present in the soluble cell extract were measured by ELISA, 24 h after induction. The low level ( - 0.2/lg MT per mg soluble protein) of MT that was observed was comparable to those reported for mouse and monkey MT production in E. coli [6,7]. These comparatively low levels of MT detected in this heterologous system may be a consequence of the inherent susceptibility of apo-MT to digestion by the

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proteolytic machinery of E. coli. Therefore, since it has been demonstrated previously that insertion of metal ions into the MT protein structure protects the holoprotein against attack by proteinases in vitro [25], the effect of adding extrinsic metal ions (Cd, Zn or Cu) on the accumulation was investigated. Firstly, however, since metal ions at high concentrations are potentially harmful to the E. coli host, it was necessary to establish conditions where sufficient metal ion could be introduced into the medium for the potential stabilisation of the recombinant protein without producing toxic effects on the cell. Initially, cadmium was examined and the effects on cell growth of its

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Fig. 3. (a) Growth of E. coli (transfected with pPWRt) in GMM-II Medium in the absence (zx) or presence of 100/IM zinc (A) or copper (11). Other conditions were as described in the legend to Fig. 2. (b) Concentration of cadmium (solid bar), zinc (hatched bar) and copper (dotted bar) in soluble extracts prepared from E. coli 1B392 LonA1 transfected with pMG 196 (left panel) or pPWRt (right panel) plasmids. The growth curves of cells containing either plasmid were identical. The cells were harvested 6.5 h after induction in GMM-II medium either alone or supplemented with Cd, Zn or Cu (at 100/tM) and washed (twice) with ice-cold GMM-II medium. Finally, the cells were resuspended in ice-cold water prior to lysis and separation of the soluble fractions for metal analysis.

182 addition (in the form of CdSO4) to the GMM-II medium are shown in Fig. 2a. Concentrations of cadmium up to 100/~M had no apparent effect on the overall growth of the E. coli cells transfected with plasmids p M G 196 or pPWRt. However, 140 /~M cadmium in the medium arrested cell growth completely. Consequently, studies on recombinant MT production were carried out with the transfected E. coli cells maintained in GMM-II medium alone or supplemented with 100 ~tM cadmium (Fig. 2b). A considerable elevation (50-60-fold) in the amount of MT present in the soluble fraction prepared from cells grown in the presence of cadmium was detected. The maximum level was reached approx. 4 h after addition of the metal ion. This level of MT detected by the ELISA remained unchanged thereafter up to 11 h (Fig. 2b). Similar experiments were performed with ZnSO 4 or CuSO 4 added to the GMM-II medium. Concentrations of 280 and 350 /~M for Zn and Cu, respectively, were found to arrest cell growth completely (data not shown). At concentrations of 100/~M in the medium, however, neither metal had a significant effect on the growth of the E. coli cells (Fig. 3a). Consequently, in order to maintain comparability with the earlier Cd experiments, each metal was added (separately) to GMM-II medium to a final concentration of 100/~M and its effect on the level of accumulation of MT was determined (Fig. 2b). In complete contrast to the results described above for Cd, no elevation in the concentration of MT in the soluble cell extracts was detected in either case over that measured in the absence of extrinsic metal (Fig. 2b). In an attempt to analyse this difference, the levels of Cd, Zn and Cu were estimated in the soluble fractions of cell extracts, following introduction of the metals (separately) at 100/~M into the GMM-II medium. After harvesting at 6.5 h post induction, in cells transfected with the (control) p M G 196 plasmid, the intracellular levels of Zn and Cu did not change appreciably (Fig. 3b; left panel) upon incubation at high extracellular concentrations. Similar results were obtained with E. coli transformed with the plasmid containing the MT insert (Fig. 3b; right panel). Cadmium could not be detected in any of the cell extracts unless the toxic metal had been introduced (deliberately) into the medium. A much higher intracellular concentration of cadmium was measured in the cells transfected with pPWRt plasmid (Fig. 3b; compare panels). Experiments were also performed with E. coli grown in L-broth. Under these conditions, it was found that a concentration of 300 /~M cadmium (or 1 mM zinc) could be tolerated without adverse effects on cell growth (data not shown). When cadmium was incorporated into the L-broth at this concentration, the level of M T expressed (500 /~g/g wet weight) was several hundred fold higher than that detected when the cells were grown in LB from which the metal was omitted (Table

TABLE I Expression of recombinant rainbow trout metallothionein in E. coli E. coli IB392 LonA1 transformed with the appropriate plasmid were grown in LB medium. The temperature was raised to 4 2 ° C to induce the plasmid and then maintained at 37 ° C for a further 30 min until the E610 = 0.4. At this stage of growth, c a d m i u m (300 # M ) or zinc (1 mM) was introduced into some cultures. Incubation was continued for a further 18 h at 37 o C and, after lysis, the level of metallothionein in the soluble cell extract, prepared as described in Materials and Methods, was determined in a competitive ELISA. Plasmid

Addition

MT (/~g/mg soluble cell protein)

Control pMG196 Insert p P W R t Insert p P W R t Insert p P W R t

None None Cd (300/~M) Zn (1 mM)

0 < 0.1 16.2 2.4

I). In the light of the earlier experiments with GMM-II medium, a substantially increased concentration of zinc was added into L-broth and its effects on the level of MT produced was determined. At this concentration (1 mM), zinc apparently was able to penetrate the cells sufficiently to enable stabilisation of MT to a level significantly above that measured in the absence of any added metal but still approx. 8-fold less than that achieved by the inclusion of Cd (at only 300/~M) in the L-broth. It was not possible to conduct experiments with Cu, since addition of this metal in substantial concentration to L-broth resulted in a heavy precipita-

T A B L E II Amino acid composition of recombinant rainbow trout metallothionein isolated from E. coli Samples were hydrolysed for 16, 40 and 72 h in vacuo at 105 ° C in 6 M HC1 following performic acid oxidation. Analyses were performed on an LKB amino acid analyser. Recoveries for Thr, Ser and Cysteic acid (Cya) were determined by extrapolation to zero time. A m i n o acid

Recombinant M T (residues per mol)

Theoretical composition from predicted sequence (residues per mol)

Cya Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Lys

16.8 4.9 3.7 9.3 2.8 2.9 6.0 2.3 1.5 0.23 * 0.2 0.4 7.0

20 4 4 10 2 3 6 2 1 1 0 0 7

* The value for methionine was obtained from analysis of a sample of protein that had not been performic acid oxidised prior to hydrolysis.

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tion of material. Table I also indicates that MT was undetectable when the parent plasmid pMG 196 (without insert) was introduced into 1B392 LonA1 and grown under the heat-shock conditions described. No cross-reactivity of the polyclonal antibody with E. coli proteins under the appropriate heat shock conditions was observed.

Purification and characterisation of recombinant M T On the basis of the above experiments, 1B392 LonA1 containing the pPWRt vector was grown in L-broth containing cadmium (300 /~M) and, after harvesting, a soluble extract was prepared as described in Materials and Methods. The concentrated extract was applied to a column of Sephadex G-75 and the cadmium-containing fractions were pooled as shown (Fig. 4a). This pooled material was buffer exchanged on a column of Sephadex G-25 and then subjected to ion-exchange chromatography on DEAE-cellulose (Fig. 4b). The major cadmium-containing fractions eluted from the column of DEAE-cellulose (i.e., fractions 14-16) were pooled for analysis. The concentration of protein in this pooled material was found (by amino acid analysis) to be 0.052 mM and the Cd concentration was 0.32 mM. This gave a calculated metal content of 6.1 g-atoms of cadmium per mole of protein. No zinc or copper was detectable in the protein preparation. Further samples of the desalted protein were subjected to performic acid oxidation prior to determination of the amino acid composition (Table II). The values obtained were in good agreement with the theoretical values for rainbow trout MT. Tyrosine, phenylalanine, tryptophan, histidine and arginine were absent, thus confirming the homogeneity of the protein. The

yield of purified recombinant MT obtained from the five litre culture was 9.4 mg. A sample of the protein preparation was stripped of cadmium (see Materials and Methods) and the cysteine residues were blocked by modification with vinylpyri-

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Fig. 5. SDS-polyacrylamide gel electrophoresis of recombinant rainbow trout metallothionein. Gels (20%) were stained with Coomassie blue after electrophoresis. Lane 1, MT after modification with vinylpyridine; lane 2, low molecular mass standards, 16.9, 14.4, 8.1, 6.2 and 2.5 kDa, respectively (Electran molecular weight markers, BDH, Poole, U.K.); lane 3, apo-MT. The origin is indicated by ( ~ ) . (Approx. 5/.tg MT was loaded).

184 dine. A sample of this material was run on a 20% SDS-polyacrylamide gel (Fig. 5) where it migrated as a single band of apparent M r 8000. The pyridyl-ethylated apo-protein (2 nmol) was subjected to N-terminal sequence analysis. Overlapping sequences were observed through 24 cycles. In cycle 1, for example, methionine (0.40 nmol) and aspartic acid (0.72 nmol) were detected, whereas in cycle two, aspartic acid (0.71 nmol) and proline (0.64 nmol) were detected. The sequence established was (Met)-Asp-Pro-Cys-Glu-Cys-Ser-Lys-Thr-Gly-Ser-CysAsn-Cys-Gly-Gly-Ser-Cys-Lys-Cys-Ser-Asn-Cys-Ala The initial yield of sequencing was 68%. This amino acid sequence matches exactly with that predicted from the gene construct used. It is clear, however, that the proteolytic enzymes of E. coli had removed much (70%) of the natural amino-terminal methionine residue of the protein. The value for methiohine recovered from sequencing (0.35 residues per mole) was in good agreement with that calculated from the amino acid analysis (Table II). Finally, the nature of the recombinant protein was investigated by examining its immunological cross-reactivity with an antiserum raised against naturally occurring rainbow trout MT. Comparisons were made by coating wells of microtitre plates with either recombia

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Fig. 6. Cross-immunoreactivity of naturally occurring and recombinant rainbow trout metallothioneins. Recombinant M T (o) and rainbow trout M T ( o ) were coated onto the wells of microtitre plates by overnight incubation at 4 ° C in 0.1 M carbonate/bicarbonate buffer (pH 9.6). (a) Samples of rainbow trout M T (between 102 and 106 pg) were incubated at 4 ° C overnight with mouse anti-trout serum (final dilution of 1/4000). Each incubation was then added to a washed, MT-coated well and allowed to incubate at room temperature for 1 h. After washing, b o u n d antibody was detected using rabbit anti-mouse IgG conjugated with horseradish peroxidase (final dilution 1/10000) and o-phenylenediamine as substrate. (b) Samples of recombinant M T were introduced as the soluble competitor.

nant or naturally occurring MT and then including natural Rainbow Trout MT (Fig. 6a) or the recombinant protein itself (Fig. 6b) as the soluble antigen in the competitive ELISA format. Similar curves were obtained in both cases. Discussion The efficient generation of proteins with a high content of -SH groups in E. coli has often been limited because of low levels of expression, a tendency to aggregate and form inclusion bodies or their toxicity. The present data have indicated that it is feasible to produce a native cysteine-rich protein with the potential to be active by affording it the opportunity to sequester metal ions in an alien environment. The production of cadmium-thionein in E. coli might thus be considered as equivalent to reconstitution of an apo-protein into its holo-form in vivo. The production of rainbow trout MT in E. coli was accurately quantified by direct measurement of the protein expressed by means of an immunoassay procedure that has been validated previously [19]. This is in contrast to previous reports [5-7] where the amount of recombinant protein produced has had to be inferred from indirect estimates based upon the addition of (radiolabelled) metal to cell extracts and the assumption of certain ratios for m e t a l / p r o t e i n binding stoichiometry. The Rainbow Trout MT synthesized in E. coli (in the presence of extrinsically added cadmium) represented approx. 1% of the soluble cellular protein. This is directly comparable to the findings reported earlier for yeast MT in E. coli [5] and is considerably higher than the level (0.05%) of expression achieved for mouse [7] or monkey [6] MT in E. coli. In the present work, high levels of MT were achieved in cells maintained in a defined (GMM-II) medium, only when cadmium was included. At comparable concentrations, neither zinc nor copper was able to emulate this effect. Among a number of possible explanations for these different responses, the simplest is that the concentrations of zinc and copper inside the E. coli cells did not attain high enough levels to be able to stabilise the apo-MT being synthesized. By contrast, a much higher concentration of cadmium was measured in the cells transfected with the plasmid containing the MT insert and indeed the ratio of C d / M T measured in the cell extracts was calculated to by approx. 7 to 1. It seems unlikely that this differential effect can be explained by a preferential binding of cadmium since, in naturally occurring MT isolated from rainbow trout, copper was shown to be the predominant metal present, despite the introduction of very high amounts of cadmium into the fish by intraperitoneal injection [26]. It is not clear whether the effect may arise from the fact that in E. coli, metal resistance often involves exclusion

185 of the metals such that copper and zinc are more actively prevented from accumulating within the cell and thus binding to apo-MT, or reciprocally whether the presence of apo-MT acts as a sink for cadmium within the cell and somehow facilitates its uptake. Since the E. coli cells carried no plasmid other than pPWRT, a plasmid-encoded cadmium efflux system [27] cannot be involved. It was suggested [7] that (low levels of) expression of a transfected mouse MT gene may confer resistance to heavy metals on E. coli, although the effect was small. By contrast, in the study of E. coli transfected with the yeast MT gene, Berka et al. [5] concluded that (much higher levels of) expression of the metal-binding protein did not produce a significant difference in metal resistance in E. coli. The findings reported here are consistent with those of Berka et al. [5] and the accumulation of a cadmium-rich Rainbow Trout MT within E. coli also agrees with their suggestion [5] that expression of a transfected MT gene does permit E. coli cells to sequester cadmium more readily from the environment. Thus, such a transfected cell might have considerable potential value in the scavenging of certain precious or toxic heavy metals from ores or wastes. Clearly, these differential effects are worthy of further investigation. The results of the present study also document thoroughly for the first time the biochemical and immunochemical characteristics of the purified recombinant Rainbow Trout MT. In this regard, it was of interest that, while the recombinant MT reflected faithfully the properties that might be expected of a metallothionein molecule, the natural N-terminal methionine residue present in this (and many other) MT(s) [1] had been largely removed during the biogenesis in E. coli. This is in total contrast to the situation observed by Berka et al. [5] for expression of Yeast MT in E. coli. In this case, whereas the eight residues at the N-terminus of the protein are apparently removed naturally in yeast itself [4], the recombinant yeast MT expressed in E. coli retained the eight amino terminal residues. The proteolytic processing machinery (for MT) in yeast and E. coli would thus appear to be different. As indicated previously, however, yeast MT has a distinctly different sequence (particularly at its Nterminus) from MTs of vertebrate species such as rainbow trout, mouse and monkey. Yeast : R. T r o u t : Monkey: Mouse:

Met-Phe-Ser-Glu-Leu-I le-Asn-PheAc-Met-Asp-Pro ......... Cys-Glu-CysAc-Met-Asp-Pro-Asn ..... Cys-Ser-Cys Ac-Met-Asp-Pro-Asn ..... Cys-Ser-Cys-

Thus, while the adequate levels of production of yeast MT observed previously in E. coli [5] may well be attributable to the different structure of the yeast protein affording protection against the proteolytic machinery of E. coli, the comparable levels of production of the vertebrate (rainbow trout) MT measured herein

would appear to be the consequence of protection against proteolysis by the relatively simple expedient of the introduction of (suitable concentrations of) cadmium into either a defined (GMM-II) medium or L-broth and its incorporation into the newly synthesized apo-protein. The levels of accumulation were much lower in the absence of added cadmium, even in the protein-deficient LonA1 strain used. Yeast MT has a free N-terminal methionine residue (see above) unlike all known vertebrate species of MT where this residue is blocked by acetylation. It would seem, however, that the acetylation is not essential for biological function, since the recombinant rainbow trout MT is not only lacking the acyl moiety but also most of the methionine residue itself. It has been suggested that this N-terminal region (and particularly the Acetyl-Met) of MT is one of the major antigenic determinants in the protein [28]. The present findings revealed a small distinction in immunoreactivity between recombinant MT (completely lacking in the acetyl moiety and largely devoid of the Met residue) and naturally occurring protein isolated from fish. Whether this small variation can be attributed to this difference in composition is clearly worthy of further investigation. However, it has also been shown [19] that while polyclonal anti-rainbow trout MT antibodies cross-react readily with MT from other species of fish, they do not react with any mammalian MTs, all of which have acetyl-Met at their N-terminus. Thus, it would seem that the N-terminal region as a whole is more likely to be the determinant rather than the acetyl-Met residue alone. Furthermore, observations made by X-ray crystallography [29] suggest that the N-terminal residues of MTs form a loose 'tail' that is not directly tied into the structure through coordination of the metal ions and may thus be readily accessible to antibodies. It is thus now feasible to produce recombinant MT in sufficiently large quantities such that structure-activity relationships may be delineated by the introduction of different metals into the protein and by alteration of the protein itself through site-directed mutagenesis. This will be the subject of further investigations.

Acknowledgements P.K. is supported by an SERC-CASE postgraduate studentship. It is a pleasure to acknowledge the contributions made to this work by Dr. T. Patel (Celltech Ltd.) [synthesis and purification of the various oligonucleotides], by Professor B.M. Dunn and Dr. B. Parten (University of Florida College of Medicine, Gainesville, FL, U.S.A.) [sequence analysis of the recombinant protein], and by our colleagues, Chris Norey, Barbara Darke and Wendy Lees [generation and characterisation of antibodies and ELISA development]. We thank Dr. A.N. Nobar for kindly supplying details of the

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composition of GMM-II medium and many valuable discussions with Dr. S. George, University of Stirling, Scotland are also gratefully acknowledged. References 1 Kagi, J.H.R. and Schaffer, A. (1988) Biochemistry 27, 8509-8511. 2 Hamer, D.H. (1986) Ann. Rev. Biochem. 55, 913-951. 3 Brown, B.A., Drozynski, Dearborn, C.B., Hadjian, R.A., Liberatore, F.A. and Haber, S.B. (1988) Anal. Biochem. 172, 22-28. 4 Wright, C.F., McKenny, K., Hamer, D.H., Byrd, J. and Winge, D.R. (1987) J. Biol. Chem. 262, 12912-12919. 5 Berka, T., Shatzman, A., Zimmerman, J., Strickler, J. and Rosenberg, M. (1988) J. Bacteriol. 170, 21-26. 6 Muraoka, Y.M. and Nagaoka, T. (1987) Appl. Environ. Microbiol. 53, 204-207. 7 Hou, Y.-M., Kim, R. and Kim, S.-H. (1988) Biochim. Biophys. Acta 951, 230-234. 8 Kay, J. and Cryer, A. (1986) NERC News J. 3, 9-10. 9 Kay, J., Brown, M.W., Cryer, A., Solbe, J.F. de E.G., Shurben, D., Garvey, J.S. and Thomas, D.G. (1987) in Metallothionein and Other Low Molecular Weight Metal Binding Proteins (Kagi, J.H.R., ed.), Experientia, Suppl. 52, pp. 627-630, Birkhauser Verlag, Basle, Switzerland. 10 Bonham, K., Zafarullah, M. and Gedamu, L. (1987) DNA 6, 519-528. 11 George, S., Leaver, M., Frerichs, N. and Burgess, D. (1989) Marine Env. Res., in press. 12 Chan, K.M., Davidson, W.S., Hew, C.L. and Fletcher, G.L. (1989) Can. J. Zool., in press. 13 Wright, E.M., Humphreys, G.O. and Yarranton, G.T. (1986) Gene 49, 311-321. 14 Messing, J. (1979) Recombinant DNA Technical Bulletin 2, 43-48. 15 Yarranton, G.T., Wright, E., Robinson, M.K. and Humphreys, G.O. (1984) Gene 28, 293-300.

16 Nobar, A.N. (1988) in Studies on Microbes Isolated from Metal Polluted Environments. Ph.D. Thesis, University of London, U.K. 17 Field, H., Yarranton, G.T. and Rees, A.R. (1988) in A Functional Recombinant Immunoglobulin Variable Domain from Polypeptides Produced in E. coli (Vaccines 88, Ginsberg, H., Brown, F., Lerner, R.A. and Chanock, R.M., eds.), pp. 29-34, Cold Spring Harbour, New York. 18 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) in Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 19 Norey, C.G., Lees, W.E., Darke, B., Stark, J.M., Baker, T.S., Cryer, A. and Kay, J. (1990) Comp. Biochem. Physiol., in press. 20 Brown, M.W., Shurben, D., Solbe, J.F. de L.G., Cryer, A. and Kay, J. (1987) Comp. Biochem. Physiol. 87C, 65-69. 21 Hirs, C.H.W. (1967) Methods Enzymol. 11, 197-199. 22 Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685. 23 Kojima, Y., Berger, C., Vallee, B.U and Kagi, J.H.R. (1976) Proc. Natl. Acad. Sci. USA 73, 3413-3417. 24 Kay, J., Cryer, A., Brown, M.W., Norey, C.G., Bremner, I., Overnell, J., Parten, B. and Dunn, B.M. (1986) Biochem. Soc. Trans. 15, 453-454. 25 Winge, D.R. and Miklossy, K.A. (1982) J. Biol. Chem. 257, 3471-3476. 26 Kay, J., Thomas, D.G., Brown, M.W., Cryer, A., Shurben, D., Solbe, J.F. de L.G. and Garvey, J.S. (1986) Environ. Health Persp. 65, 133-139. 27 Silver, S. (1983) in Biomineralisation and Biological Metal Accumulation (Westbroek, P, and De Jong, E.W., eds.), pp. 439-457, D. Reidel Publishing co., Dordrecht. 28 Winge, D.R. and Garvey, J.S. (1983) Proc. Natl. Acad. Sci. USA 80, 2472-2476. 29 Furey, W.F., Robbins, A.H., Clancy, L.L., Winge, D.R., Wang, B.C. and Stout, C.D. (1986) Science 231,704-710.