THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

VoI. 261, No. 12, Issue of April 25, pp. 5658-5662.1986 Printed in U.S.A.

Overproduction and Purification of the B2 Subunit of Ribonucleotide Reductase from Escherichia coZi*

(Received for publication, July 22, 1985)

Britt-Marie SjobergSB, Solveig Hahne@, Margareta KarlssonSB, Hans Jornvallll, Mikael GoranssonII , and Bernt Eric UhlinII From the $Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center S-751 24 Uppsala, $The Medical Nobel Institute, Department of Biochemistry I, and the VDepartment of Chemistry, Karolinska Institute, S-104 01 Stockholm, and the 11 Department of Microbiology, University of Umeti, S-901 87 Umei;, Sweden

The nrdB gene of Escherichia coli, coding for the B2 protein of ribonucleotide reductase, has been cloned in a runaway-replication vector. The runaway derivative pBEU17 carries the promoter-proximal portion of the E. coli alanyl-tRNA synthetase gene and proved useful for expressing cloned genes lacking their native tran- scription initiation signals. The alas promoter is lo- cated approximately 500 base pairs upstream of a sin- gle BarnHI restriction endonuclease cleavage site uti- lized in the construction of an expression recombinant plasmid, pBS1, for the nrdB product. After 5-h ther- mal induction of cells carrying the runaway recombi- nant pBS1, protein B2 constituted 40% of the soluble protein fraction of the cells. The high concentration of protein B2 in crude extracts of induced cells has ena- bled a simplified purification scheme to be developed for production of homogeneous and concentrated B2 preparations. Protein B2 produced from pBSl is iden- tical to the chromosomally encoded nrdB product of E. coli as regards molecular mass on sodium dodecyl sul- fate-polyacrylamide gel electrophoresis, enzyme activ- ity, tyrosine radical content, and structure of the bi- nuclear iron center. Amino acid sequence analysis showed that the two polypeptide chains of protein B2 are identical. They start with an alanine residue, and the first 30 residues confirmed the amino acid sequence predicted from the nucleotide sequence of the nrdB gene, apart from an NH2-terminal processing removal of the initiator methionine.

Ribonucleotide reductase is an essential component of all cells. The enzyme catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides and thereby pro- vides a balanced supply of all four precursors for DNA syn- thesis (1). The prototype of all known eukaryotic ribonucle- otide reductases, as exemplified by the Escherichia coli en- zyme, consists of two non-identical subunits. In E. coli they have been denoted proteins B1 and B2. The B2 subunit contains a unique prosthetic group consisting of a tyrosine radical stabilized by an adjacent binuclear iron center (2). During the last decade, the interest in ribonucleotide reduc- tase has taken the form of biophysical (3, 4), structural ( 5 ) , and mechanistic studies (6), techniques which demand large

* This work was supported by grants from the Swedish Medical and Natural Science Research Councils and the Magn. Bergvall Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

quantities of concentrated, homogeneous preparations of en- zyme.

Cloning of the E. coli genes coding for ribonucleotide re- ductase, the nrdA and B genes, was first achieved in a defec- tive, heat-inducible X vector (7) and later in the multicopy plasmid pBR322 (8). Efficient expression of ribonucleotide reductase was found in both of the two recombinant con- structs, and the levels of proteins Bl and B2 reached a few per cent in the cytosolic protein fraction of the crude extracts. For our present structural and mechanistic studies on ribo- nucleotide reductase, we desired a system efficiently express- ing each subunit separately.

High level expression of cloned products in bacterial cells is best achieved in systems with controllable transcription. For overproduction of protein B2, we have combined the nrdB structural gene with a potent promoter in a runaway-replica- tion plasmid vector. The runaway replicon, which originates from the plasmid R1 (9), can be thermally induced to amplify itself in growing cells, and the result is a highly increased gene dosage (10, 11). We here show that the promoter-proxi- mal portion of the E. coli alanyl-tRNA synthetase gene (alas, Refs. 12 and 13) can be utilized for expression of cloned genes in this vector. Upon induction, the B2 subunit of ribonucleo- tide reductase constituted about 40% of the soluble protein fraction in a crude extract. This overproduction simplified the purification of the protein.

EXPERIMENTAL PROCEDURES

Materiak-Restriction endonucleases BamHI, EcoRI, and SalI were from Boehringer Mannheim GmbH or New England Biolabs and were used according to directions from the manufacturers. Bac- teriophage T4 DNA ligase was a kind gift from Dr. G. Magnusson, Department of Virology, Uppsala University, Uppsala, Sweden. E. coli protein B1 and thioredoxin reductase were purified as described earlier (14, 15). E. coli thioredoxin was a kind gift from Dr. A. Holmgren, Department of Chemistry, Karolinska Institute, Stock- holm, Sweden.

Bacterial Strains a d Plasmids-Plasmids were generally main- tained in E. coli C600 thr-1 leu-6 thi-1 supE44 lacy1 gal pro rpsL hsr- hsm+ (16). Lac+ recombinants were selected in MC1061 araD139 A(ara leu) 7697 AlacX74 galU- galK- hsr- hsmf rpsL (17), and nrdB- carrying plasmids were selected in KK826 ualR A(lae pro) his cdd nalA nrdB glpT thi rpsL recA, which was a kind gift from Dr. 0. Karlstrom, Department of Microbiology, University of Copenhagen, Copenhagen, Denmark. Plasmid pBEU17 (see “Results”) is an am- picillin-resistant derivative of pKN402 carrying a DNA fragment from the E. coli recA-alas region (11). Plasmid pMC903 (18) was used as a source of a BamHI fragment containing the lacZYA operon in the construction of plasmids pBEU6O (see “Results”) and pHMG2 (19). PlasmidpPSS is a tetracycline-resistant recombinant of pBR322 carrying both structural genes of E. coli ribonucleotide reductase (nrdA nrdB) (8).

Media and Growth Conditiom-Cells were usually grown in LB

5658

Overproduction of the B2 Subunit of Ribonucleotide Reductase 5659

broth (20) a t 30 "C. For the hydroxyurea selection, transformants were plated at 30 or 34 'C on minimal agar plates (21) supplemented with threonine (50 pg/liter), leucine (80 pgfliter), thiamine (1 pg/ liter), and hydroxyurea (0.1-4 mg/rnl). For large-scale cultivation of CSOO/pBSl, cells were grown a t 30 "C in 17 liters of SLBH-medium (22) in a New Bmnswick MF-128s fermentor equipped with auto- matic pH control. When the culture reached a density of -1 X lo* cells/ml, the temperature was raised to 42 "C. Cells continued to grow, as monitored by an increase in optical density a t 640 nm, for approximately 3-4 h, a t which time the culture was rapidly cooled to 0 "C by mixing with crushed ice. About 100 g of cells were harvested by continuous centrifugation in a Sharples centrifuge and stored at -70 "C.

Molecular Cloning-Procedures were according to Maniatis et al. (23).

For insertion of the lac2 gene downstream of the alas promoter, BamHI-digested pBEU17 DNA was mixed with BamHI-digested DNA of the h c fusion vector pMC903 (18). After ligation, the DNA was used for transformation of the lac deletion strain MC1061, and ampicillin-resistant Lac+ clones were screened on MacConkey indi- cator plates a t 30 "C.

For cloning of the nrdB gene of E. coli, 10 pg of pPS2 DNA and 30 ,ug of pBEU17 DNA were cleaved with an excess of BamHI endonu- clease at 37 "C for 18 h. The mixture was extracted with phenol and concentrated by ethanol precipitation prior to ligation for 22 h at 15 "C and at a DNA concentration of about 100 pg/ml. Approximately one third of the ligation mixture was used to transform 2 ml of competent KK826 cells. Aliquots of 0.2 ml were then plated in minimal soft agar on minimal plates containing varying amounts of hydroxyurea. The plates were incubated for 3 days at 30 or 34 "C.

Assay for @-Galactosidase Activity-The specific activity of P-galac- tosidase in bacteria carrying lacZ fusion plasmids was determined according to Miller (24).

Assay for Protein B2 Activity-The enzymatic activity of protein B2 was determined spectrophotometrically (14) with an excess of protein B1 (1-2 p ~ ) , thioredoxin (13 p ~ ) , and thioredoxin reductase (0.7 p ~ ) . ATP (1.5 mM) or dTTP (40 p ~ ) was used as effector and CDP (0.5 mM) as substrate.

Protein Determination-Preparations were routinely followed by UV absorbance a t 280 nm. A molar extinction coefficient (Eao) of 117,000 M" cm" was used for protein B2 (3, 25). Protein content in crude extracts was determined by the method of Lowry (26) or with the Bradford reagent (27).

Rocket Immune Electrophoresis-Crude extracts were prepared from -1.5 X 10" cells by a cleared lysate procedure as described before (16). Supernatants were used in proper dilutions for rocket immune electrophoresis (7) with three different amounts of homoge- neous protein B2 as standard.

Denaturing Gel Electrophoresis-Aliquots of 1 ml were withdrawn from growing cultures, quickly cooled, and centrifuged in Eppendorf tubes at 4 "C. The pellet was resuspended at a density of 1.5 X 10" cells/ml in minimal medium. For SDS1-polyacrylamide gel electro- phoresis (28), 5 g1 of the cell suspension were diluted into 45 pl of sample buffer and boiled for 2 min. Each slot was loaded with 20 pl of boiled extract. A mixture of proteins B1 and B2 (about 2 pg each) was used as a reference.

Large-scale Purification of Protein B2-All manipulations were at 4 "C. The first steps in the purification scheme were essentially as described before for proteins B1 and B2 (14), except that the extrac- tion buffer was 50 mM Tris-C1, pH 7.6, containing 20% glycerol and 1 mM phenylmethylsulfonyl fluoride (buffer A). Briefly, 66 g of heat- induced C600/pBS1 cells were disintegrated and extracted in buffer A. Nucleic acids were removed by streptomycin precipitation. The protein fraction was concentrated by ammonium sulfate precipitation and dissolved in a small volume of buffer A. At this stage, the preparation was divided into three equal fractions which were stored at -70 "C and worked up separately. Each fraction was desalted by passage through a column of Sephadex G-25 equilibrated with buffer A and applied to a column of DEAE-cellulose (approximately 1 mg of protein/ml of cellulose) previously equilibrated with 0.15 M potas- sium phosphate buffer, pH 7.0, containing 10 pM phenylmethylsul- fonyl fluoride. Protein was eluted with a gradient (10-volume excess over cellulose) from 0.15 to 0.30 M phosphate. Protein B2 eluted as a prominent peak at approximately 0.22 M phosphate. B2-containing fractions were pooled, concentrated about 400 times to approximately

The abbreviation used is: SDS, sodium dodecyl sulfate.

E P P E PPSP P E K

pBEU17 & Am$ &,

E P P E P PPSP P E KB E

pBEU60 1 Amp'

PPSZ p- - nrdA nrdB TetR

B P P E P PPSP P E K E K P S

PBSl

1 1 1 - 1 1 l l l l I 1 1 1 1 1 l I I L ~ I

5 i o 15 20 2Skb

FIG. 1. Plasmid constructions. Restriction endonuclease sites indicated B, BamHI; E, EcoRI; K, KpnI; P, PstI; and S, SalI. Origin of DNA fragments shown: E. coli DNA, open bar; plasmid R1, hatched bar; and plasmid pBR322, filled bar. The horizontal arrows show direction of transcription for the alas and nrd genes.

1.3 ml by ultradialysis against buffer A overnight, and stored at -70 "C. The three concentrated DEAE fractions were pooled (4 ml) and applied to an ultrogel AcA34 column (2.5 X 120 cm) and eluted with a modified buffer A containing only 10 pM phenylmethylsulfonyl fluoride. SDS-polyacrylamide gel electrophoresis was used to monitor the purity of fractions before they were pooled and concentrated by ultradialysis against buffer A without phenylmethylsulfonyl fluoride. Concentrated protein B2 was stored in aliquots of 0.25 ml at -70 "C.

Amino Acid Sequence Determinution-Homogeneous protein B2 (50 nmol) was reduced with dithiothreitol and carboxymethylated with i~do['~C]acetate (25). The amino acid sequence was determined by liquid-phase sequencer degradation (Beckman 890D) using a 0.1 M Quadrol peptide program and application into glycine-precycled Polybrene (29). Phenylthiohydantoin derivatives were identified by high-performance liquid chromatography (30) coupled, when appli- cable, with thk-layer chromatography and radioactivity measure- ments (29). The repetitive yield was 96%.

RESULTS

A Runaway Vector Containing the alas Promoter-Genes nrdA and nrdB encoding the ribonucleotide reductase sub- units B1 and B2 are transcribed as an operon in which nrdA is the promoter-proximal gene (31). To optimize production of the promoter-distal gene product, the B2 protein, we needed a vector which would simultaneously promote nrdB gene transcription and allow amplifiable expression. The derivative pBEU17 of the thermoinducible runaway replicon pKN402 (10) was chosen for this purpose. Plasmid pBEU17 is a derivative of the runaway plasmid pBEU14, which carries a 3-kilobase fragment of the E. coli chromosome including the recA gene (11).2 Due to an EcoRI deletion of pBEU14, only the COOH-terminal coding portion of recA and a 1.4-kilobase region downstream to the nearest BamHI site of the cloned chromosomal DNA were retained in pBEU17. Since the alas gene of E. coli is encoded in this region, we predicted that transcription from the alas promoter would proceed through the single BamHI-site in plasmid pBEU17 (Fig. 1). To test if it would be of potential value for expression of cloned genes, we introduced a BanHI fragment carrying the lacZYA operon (without its promoter) into the BanHI site. The Lac+ clone pBEU6O carried the lac operon fragment inserted in the orientation which should allow transcription of lacZYA from the alas promoter (Fig. 1). The relative strength of the alas- promoted transcription was shown when the expression at 30 "C of p-galactosidase from pBEU60 was compared to that of plasmids containing tet or the native lac promoters (Table I). Despite the fact that the latter two plasmids are pBR322 constructs and thereby have higher copy number than the pBEU17 replicon at 30 "C, the alas-lac fusion hybrid produced

B. E. Uhlin and A. J. Clark, unpublished experiments.

5660 Overproduction of the B2 Subunit of Ribonucleotide Reductase

TABLE I Characterization of ahS-Dromoted transcriDtion

Plasmid" Promoter controlling locZ activity a t

30 'C

pBEU6O a h S pHMG2 tet pSKS106 lac

units 10,910 9,623 3,060

' Plasmids were maintained in strain MC1061.

C600/pBEU17 C600/pBSI

0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 %

Bl J

B2 I

gB1

sB2

FIG. 2. SDS-polyacrylamide gel electrophoresis of lysed cells of C600/pBEU17 and C6OO/pBS1 harvested at different times after heat induction. C600/pBEU17 grown in LB medium (20) ,and harvested 0-6 h after heat induction is shown by the top values, as is C6OO/pBSl grown in SLBH medium (22) and harvested 0-8 h after heat induction.

the highest level of P-galactosidase. Overproduction of the E. coli nrdB Gene Product-The

efficient alas-promoted transcription of pBEU17 was further utilized for insertion of a 6.4-kilobase DNA fragment carrying the nrdB gene of pPS2 into the BamHI site of pBEU17, as outlined in Fig. 1. Recombinants carrying the nrdB gene were easily scored as hydroxyurea-resistant transformants of the recipient strain KK826, which is sensitive to the drug because of its nrdB lesion. One isolate, carrying a plasmid denoted pBS1, was resistant to 4 mg/ml hydroxyurea, i.e. a 50-fold increased level of resistance as compared to the recipient strain. Analysis with restriction endonucleases confirmed that plasmid pBSl carries the nrdB gene in a position which allows transcription from the alas promoter (Fig. 1). It should be noted that the hybrid plasmid also carries most of the pBR322 replicon and may therefore use either of the vector replicon functions at 30 "C. Studies with E. coli C600 cells carrying pBSl confirmed that the plasmid mediated synthesis of the B2 protein. The production of nrdB protein was monitored by rocket immune electrophoresis and by SDS-polyacrylam- ide gel electrophoresis of total cellular protein extracts. As shown in Fig. 2, a temperature shift from 30 to 42 "C, allowing for runaway replication, resulted in accumulation of the B2 protein to such an extent that it became the predominant protein and was easily detected on stained polyacrylamide gels. The nrdB gene product, as determined by rocket immune electrophoresis, represented up to 40% of the total soluble protein of the cellular extract after 4-5 h at the post-shift temperature (Table 11). This gene product amplification cor- responds to an approximately 500-fold increase compared to the normal cellular content of the protein in E. coli (Ref. 7 and Table 11). Even before heat induction, pBS1-carrying

TABLE I1 Levels of protein B2 in crude lysates of C600 cells carrying

recombinant plasmids

Plasmid Growth conditions Protein B2 content'

gglmg protein pBEU17 30 "C 0.3

43 "C, 4 h 0.3 pBSl 30 "C 17

43 "C, 2 h 100 3 h 170 4 h 240 5 h 400

' Determined by rocket immune electrophoresis.

cells contained clearly elevated levels of immunoreactive nrdB protein, which was about 2% of the cytosolic fraction (Table 11). For comparison it should be mentioned that at 30 "C the amount of the chromosomally encoded B1 subunit (i.e. the nrdA gene product) in cells of strains C600/pBEU17 and C600/pBS1 was 0.9 and 1.4 pg/mg of protein, respectively. The level of the B1 protein did not change significantly during the temperature shift.

Purification and Characterization of Protein B2 from Strain C600/pBSl"The overproduction obtained with plasmid pBSl allowed simplification of the purification procedure for protein B2. A large-scale preparation was carried out with 66 g of C6OO/pBSl cells after a temperature-shift amplification experiment. The purification is summarized in Table 111. All purification steps were performed in the presence of phenyl- methylsulfonyl fluoride to prevent proteolytic degradation. Before the DEAE chromatography step, the purification pro- cedure was essentially the same as reported earlier for the recombinant X lysogen (7, 14) and resulted in a severalfold purification of protein B2 without loss of activity. The main purification was achieved by chromatography on DEAE-cel- lulose (Table 111), from which protein B2 was eluted as the major protein peak. B2 was essentially pure at this stage, but the last exclusion chromatography step was included in order to remove some minor impurities. The purified B2 protein had a mobility corresponding to a molecular mass of 43.5 kDa and appeared homogeneous on SDS-polyacrylamide gel elec- trophoresis. The final preparation had a specific activity of 5880 units/mg of protein. It was identical to the chromo- somally encoded B2 protein also in tyrosyl radical and struc- ture of binuclear iron center (data not shown).

The B2 preparation from C600/pBS1 was submitted to NH2-terminal amino acid sequence analysis. The sequence of the first 30 residues was Ala-Tyr-Thr-Thr-Phe-Ser-Gln-Thr- Lys-Asn-Asp-Gln-Leu-Lys-Glu-Pro-Met-Phe-Phe-Gly-Gln- Pro-Val-Asn-Val-Ala-Arg-Tyr-Asp-Gln, which confirmed the amino acid sequence predicted from the DNA sequence of the nrdB gene (31). Furthermore, the results show that the two polypeptide chains of B2 are identical and that the initiator methionine is removed from the native protein, which starts with the subsequent alanine residue.

DISCUSSION

Previous cloning of the B2 structural gene in pBR322 resulted in low expression of B2 protein when nrdB was separated from the promoter-proximal nrdA gene (8). We have now shown that cloning of nrdB in a runaway replicon results in a large overproduction of the B2 subunit of E. coli ribonucleotide reductase, explained by the fact that the am- plifiable vector pBEU17 carries the potent alas promoter of E. coli.

The B2 protein obtained by the present procedure appears

Overproduction of the B2 Subunit of Ribonucleotide Reductase 5661

TABLE I11 Purification scheme of protein B2 from 66 g of C6OO/pBSl cells harvested 4 h after heat induction

Fraction Volume Protein Enzyme Specific activity activity

ml mg U n i t S / X l @ % -fold units/

Yield" Purification"

mg Crude extract 244 11,700 2.05' Streptomycin sulfate and am- 65 3,190 4.95 1,490 100

DEAJ? chromatography Ultrogel AcA34 chromatography 2.0 387 2.29 5,880 46 14

Estimated only in last step, because of explanations in Footnotes b and c. The purification was derived using

' The enzymatic assay is inaccurate in crude extracts because of the presence of contaminating nucleotides,

e Because of the presence of phosphate, this fraction was assayed with dTTP as allosteric effector, which gives

monium sulfate precipitation 4.0 398 2.16" (5,430)

the protein content of the first step and the enzyme activity of the second step as initial values.

nucleotide-degrading enzymes, and other NADPH-consuming enzyme systems (14).

lower values than with ATP as allosteric effector (14).

identical in tyrosine radical content, iron content, specific activity, and subunit molecular mass to the B2 protein puri- fied from thymine-starved E. coli B3 (25) and B2 from the X- lysogenic overproducer KK546 (7). Its amino acid composition is similar to that obtained earlier for B2 from E. coli B3 cells (25) and to that deduced from the DNA sequence (31) of the nrdB gene (data not shown). Moreover, the NHP-terminal amino acid sequence (IV) is identical to residues 2-31 of the DNA-derived amino acid sequence of protein B2 (31). It is also clear from the analysis that, despite the hybrid operon construction in pBS1, nrdB gene translation starts at the normal initiation signal. There was no indication of other sequences (e.g. due to read-through translation from alas or other sequences) at the 5'-end of nrdB. This is consistent with the way the hybrid was constructed. Presumably, any translation initiated at the alas start codon should terminate at the nrdA gene stop codon located upstream of the nrdB gene within the BamHI fragment used. According to the DNA sequence data, the reading frames of alas and nrdA are in register at the BamHI junction point (31, 32). The predicted alas-nrdA fusion protein would have a molecular mass of about 38 kDa. It was not possible to distinguish such a polypeptide band on stained gels. A fusion protein of this type probably has an aberrant conformation and may be rapidly degraded by E. coli proteases.

In the pBEU17 derivative pBS1, the relatively high level of nrdB expression at permissive temperature (Table 11) implies that transcription from alas is largely derepressed. Amino- acyl-tRNA synthetases are known to be regulated in two separate ways: derepression occurring during restriction of the cognate amino acid and metabolic derepression as a func- tion of the cellular growth rate (33). Autoregulation of the alas operon occurs by binding of the synthetase to a palin- dromic operator sequence close to the alas promoter. I n vitro studies have shown that there is virtually no autorepression of the alas operon by alanyl-tRNA synthetase at its observed intracellular concentration (13). Instead in vivo derepression is believed to be mediated by the alanine-charged synthetase which has a lower binding constant for the operator sequence. In experiments with strain C600/pBS1, we have not seen any effect on expression of protein B2 by excluding alanine from the growth medium (data not shown). In studies with the alas gene cloned on pBR322, the amount of alanyl-tRNA synthe- tase was found to be amplified about 10-fold (12). There is evidently a clear effect of gene dosage when the alas gene is cloned on multicopy plasmids. The estimated amounts of alanyl-tRNA synthetase (0.4 PM or approximately 250 mole- cules/cell) and alanine (1-5 mM) in E. coli cells are well above the copy number of such plasmids. Perhaps the regulatory

circuits of the alas operon act less efficiently on circular DNA molecules in vivo, resulting in a lack of autogenous repression of the nature observed with linear DNA fragments in vitro (13). Another possibility is that the stability of the mRNA from our hybrids is different from that of the native alas transcript. Evidently, the alas promoter can be employed as an alternative to the more commonly used promoters in expression plasmid vectors (Table I). In combination with the plasmid-mediated gene dosage effect, an efficient expression of protein B2 was obtained with C6OO/pBSl at the inducing temperature.

The present overproduction has simplified the procedure for purification of protein B2 and thereby aided the structural and functional studies of ribonucleotide reductase. Crystals suitable for x-ray work were recently obtained of protein B2 prepared from strain C600/pBSl (5). Protein B1, the other subunit of ribonucleotide reductase, has separately been sub- ject to efficient overproduction in a similar manner (34). The nrdA gene was cloned by insertion of a KpnI fragment in the unique site of pBEU17. In that case, the native nrd promoter was included in the recombinant. Upon thermoinduction there was a 200-fold overproduction of protein B1 as compared with wild-type cells (34).

The plasmid pBEU17 has also been used for overproduction of the products of E. coli dnaA, dnaZ, and ssb genes (35). In this case, the structural genes presumably carried their own promoters, since potent expression was obtained even though the cloning rationale involved a site upstream of the alas promoter or a deletion of a fragment of pBEU17 comprising the alas promoter. However, for construction of the ssb deriv- ative, the KpnI site downstream of the alas promoter was used. This construct resulted in 60 times elevated level of the ssb product (35). Since the direction of transcription of the ssb gene was not determined in that experiment, it cannot be concluded whether or not expression of ssb benefited from the fact that the alas promoter is present in the vector. However, in this context we would like to point out that our present studies show that it may not be necessary to separate the DNA fragment of interest from the replicon of a conventional vector (e.g. pBR322) when subcloning into the runaway vector is carried out. The double replicon construct pBSl retained the runaway-replication behavior typical of the vector pBEU17 itself. Furthermore, the relatively large size (20 kilobases) of the hybrid did not appear to limit the overpro- duction. In cases where the fragment containing a particular gene does not allow for direct selection or when convenient restriction sites are lacking, the fusion of entire plasmids offers a simple strategy toward increasing the gene dosage by runaway replication. Cloning of the nrdA (34) and the nrdB

5662 Overproduction of the B2 Subunit of Ribonucleotide Reductase

1. 2.

3.

4.

5.

6.

7.

8. 9.

10.

11. 12.

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