THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 19, Issue of July 5, pp. 10988-10992, 1990 Printed in U. S. A.

Expression and Secretion of Mirabilis Antiviral Protein in Escherichia coli and Its Inhibition of in Vitro Eukaryotic and Prokaryotic Protein Synthesis*

(Received for publication, December 15, 1989)

Noriyuki Habuka, Kiyotaka Akiyama, Hideaki Tsuge, Masashi Miyano, Takashi Matsumoto, and Masana Noma From the Life Science Research Laboratory, Japan Tobacco, Inc., 6-2 Umegaoka, Midori-ky Yokohama, Kanagawa 227, Japan

Mirabilis antiviral protein (MAP), a ribosome-inac- tivating protein, exhibits inhibitory effects on both plant virus infection and protein synthesis. To study these functions by site-specific mutagenesis, the total synthetic gene of MAP was constructed and expressed in Escherichia coli. However, the growth of the host was inhibited by the products, and the yield of MAP was very low. To improve the system for expressing MAP, an expression vector, pSH7, was constructed. This vector is based on the high copy number plasmid pUC19 and includes PL promoter and temperature- sensitive ~1857 repressor. The plasmid also contains the ompA signal sequence and the total synthetic MAP gene. The MAP gene was expressed and its product was secreted into the culture medium after E. coli transformants were cultivated at 30 ‘C and the tem- perature was raised to 42 “C. The secreted MAP was then purified and characterized. This protein was iden- tical to native MAP as determined by its mobility in sodium dodecyl sulfate-polyacrylamide gel electropho- resis, the amino acid sequence at the NH2 terminus, and its inhibitory effect on in vitro protein synthesis. MAP was found to inhibit the in vitro protein synthesis of rabbit reticulocyte and wheat germ. It further showed an ICso concentration of approximately 200 nM in an E. coli in vitro translation system in contrast to ricin A-chain, a well known ribosome-inactivating protein.

Mirubilis antiviral protein (MAP)’ isolated from the roots of Mirubilis jalapa L. inhibits the mechanical transmission of plant viruses, such as tobacco mosaic virus (1, 32, 33). In addition, it inhibits the in vitro protein synthesis of mam- malian and plant systems (2).

Several proteins that inhibit in vitro protein synthesis have been isolated from a variety of plants, e.g. ricin from Ricinus communis (3), tritin from Triticum aestivum (4), pokeweed antiviral protein from Phytolucca americanu (5), and from microorganisms, e.g. Shiga toxin from Shigella dysenteriue 1 (6). It has been suggested that protein synthesis inhibition by ricin A-chain, the catalytic subunit of the toxin, is caused by its RNA N-glycosidase activity (7,8); that is, it cleaves an N- glycosidic bond at A4324 of rat liver 28 S rRNA, which resulted in the inactivation of the ribosomes. This RNA N-glycosidase

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The abbreviations used are: MAP, Mirabilis antiviral protein; RIP, ribosome-inactivating protein; SDS, sodium dodecyl sulfate.

activity has also been found in other proteins, such as tritin and Shiga toxin (9). Therefore, these proteins are called ribosome-inactivating proteins (RIP) (9, 10).

We found that MAP consists of a single peptide without sugar moiety (32, 33), and we also determined the complete amino acid sequence consisting of 250 amino acids (2). In addition, we found 24% sequence homology and some mean- ingful conservation between MAP and ricin A-chain se- quences: i.e. arginine residues, which have been suggested to play an important role in the inhibition of protein synthesis (ll), and two highly conserved regions, one of which contains 11, and the other, 13 residues (2). These observations suggest that the inhibitory mechanism of MAP may be similar to that of ricin A-chain (2). We further synthesized the total MAP gene containing 12 unique restriction sites, averaging 65 base pairs apart, for expression in Escherichia coli and in vitro mutagenesis (2). When the gene was expressed under control of the tat promoter in E. coli, it produced a protein that had the same mobility as native MAP on SDS-polyacrylamide gel electrophoresis and cross-reacted with antibody against MAP (2). Its yield was estimated to be only 3.6 mg/liter (2). The main cause of the low yield seemed to be the effects of the product on the growth of the host (2). On the other hand, the expression of ricin A-chain gene has been reported to give a good yield of the product in E. coli. (12, 13). We think that these differences arise from their different toxicities to E. coli. So far, there have been no reports on the inhibitory effect of RIPS on prokaryote.

In this paper, we describe the expression of MAP gene, and the substantial production of MAP, which injured E. coli as a host, and then the inhibitory effect of MAP on eukaryotic and prokaryotic protein synthesis and a comparison with ricin A-chain.

EXPERIMENTAL PROCEDURES

Materials

N,N-Diisopropylphosphoramidites and the other reagents for oligonucleotide synthesis were purchased from Applied Biosystems Japan (Tokyo, Japan). DNA polymerase Klenow fragment, T4 DNA polymerase, T4 DNA ligase, T4 nucleotide kinase, and the restriction enzymes PvuI and Ban111 were from Toyobo Co., Ltd. (Osaka, Japan). The other restriction enzymes were from Nippon Gene Co., Ltd. (Toyama, Japan). Carboxymethyl-Sepharose and Blue-Sepharose were from Pharmacia Japan (Tokyo, Japan). Rabbit reticulocyte lysate, wheat germ extract, amino acid mixture, and L-[35S]methio- nine were from Amersham Japan Co., Ltd. (Tokyo, Japan). Ricin A-chain was purchased from Vector Laboratories, Inc. Native MAP purified from Mirabilis plant (2) was kindly

10988

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Expression and Secretion of MAP by E. cob 10989

donated by Masatoshi Arita (Sea Water Science Research Laboratory, Kanagawa, Japan).

Bacterial Strains and Plasmids

E. coli strains HBlOl (F-, hsd20 (r-, m+), recAl3, ara-14, proA2, lacY1, galK2, rpsL20 (Sm’), ~~1-5, mtl-1, supE44, X-) and NQQcI’ (thi, supE44, gyrA96, endAl, hsdR17 (r-, mf), retAl, A(lac-proAB), AF’ episome-traD36, proAB, lacP2 M15) were used for cloning experiments. E. coli strain MM294 (F-, e&Al, hsdR17 (r-, m+), supE44, thi-1) was used for expression work. The E. coli strains Ql3 (Hfr; met, ma, pnp, tyr) used for the preparation of the protein synthesis system and P4X8 (HfrP4X8, metBX-) used as a host for MS2 phage were kindly provided by National Institute of Genetics, Japan. The E. coli RNA phage MS2 used for the preparation of mRNA was obtained from Institute for Fermentation, Osaka. The plasmids pKK223-3 and pPL-X were purchased from Pharmacia Japan (Tokyo, Japan). pUC19, pHSG397 and X phage DNA were from Takara Shuzo Co., Ltd. (Kyoto, Japan).

Methods

Construction of MAP Expression Vector pSH7-The expression and secretion vector pSH7 is shown in Fig. 1. DNA linker I (Fig. 2a) was prepared with a DNA synthesizer (Applied Biosystems Japan 381B) using a phosphoramidite method (14). The linker contains restriction sites, the Shine- Dalgarno sequence, and the NH?-terminal sequence of MAP to facilitate the insertion of the total synthetic MAP gene (2) and its expression. The complementary DNA fragments were kinated and annealed to make double-stranded DNA as de- scribed in a previous paper (2). The plasmid pKS2 was ob- tained by insertion of DNA linker I into the EcoRI-Hid111 site of pKK223-3.

The plasmid pKS2 was digested by BamHI, filled up by DNA polymerase Klenow fragment, and then digested by PuuI. The insertion of a pUC19 PuuI-PuuII fragment con-

NdeI,,SalI

FIG. 1. Expression and secretion vector of MAP, pSH7.

FIG. 2. Synthetic DNA linkers. a, DNA linker I; b, DNA linker II.

taining the replication origin into the pKS2 fragment yielded the high copy number plasmid pKS3.

A fragment containing PL promoter sequence was obtained by BomHI-HpaI digestion of the plasmid pPL-X. The plasmid pKS3 was digested by PstI, and its protruding 3’-end was deleted by T4 DNA polymerase and then digested by BamHI. The insertion of this PL promoter fragment into the fragment of pKS3 yielded the plasmid pSH4.

The ~1857 repressor sequence was cloned by insertion of a BglII-BanIII-digested fragment of X phage DNA into a BamHI-Ban111 site of plasmid pHSG397. A DNA fragment with the ~I857 repressor sequence was obtained by XhoI- Ban111 digestion of the inserted plasmid and filled up by DNA polymerase Klenow fragment. The plasmid pSH4 was di- gested by BamHl and filled up by the Klenow fragment. The insertion of the ~1857 repressor sequence into the fragment of pSH4 yielded the plasmid pSH5.

Pre-MAP gene (the total synthetic MAP gene without an NH2 terminus) was obtained by XbaI-Ban111 digestion of the plasmid pMH1 (2). The insertion of the pre-MAP gene into the XbaI-Ban111 site of pSH5, which had the NHp-terminal sequence of MAP, yielded the expression vector pSH6.

To construct a MAP secretion vector, DNA linker II (Fig. 2b) was prepared. The linker coded the ompA signal sequence (15) and the NHz-terminal sequence of native MAP between the NdeI and XbaI sites. The insertion of the linker II at the NdeI-XbaI site of pSH6 yielded the MAP secretion vector pSH7 (Fig. 1).

Expression of the MAP Gene in E. coli-E. coli transform- ants (MM294) grew in L-broth containing 50 pg/ml ampicillin at 30 “C. When its absorbance at 550 nm reached 0.8, expres- sion was initiated by increasing the culture temperature to 42 “C! through the addition of the same volume of the medium at 55 “C. MAP production was achieved by continuous aerobic incubation at 42 “C. MAP in the cytosol and the periplasmic space of E. coli was fractionated by the osmotic shock method (16) after the cells were removed from the culture medium by centrifugation. P-Lactamase was assayed at pH 7.0 by the method described by Sawai et al. (17). One unit of /3-lactamase was defined as the amount that hydrolyzed 1 pmol of ampi- cillin/min at 30 “C.

Purification of Secreted MAP-E. coli transformant (MM294/pSH7) bodies were removed from the culture me- dium by centrifugation. Two liters of the medium was adjusted to a 90% saturation of ammonium sulfate. After centrifuga- tion (10,000 X g, 20 min), the resulting precipitate was dis- solved in 40 ml of Buffer A (10 mM sodium phosphate, pH 6.0) and dialyzed against the same buffer. The solution was applied to a carboxymethyl-Sepharose column (26 X 40 mm). In each chromatograph, the effluent stream was monitored for absorbance at 280 nm, and the concentration of MAP was measured by enzyme-linked immunosorbent assay using the antiserum against MAP (2). After the column was washed with Buffer A, MAP was eluted with a 200-ml linear gradient of sodium chloride (O-O.5 M) in Buffer A. Fractions containing MAP were collected and dialyzed against Buffer B (10 mM Tris-HCl, pH 8.0). Then the solution was applied to a Blue- Sepharose column (5 x 50 mm). After the column was washed with Buffer B, MAP was eluted with a lo-ml linear gradient of sodium chloride (O-O.2 M) in Buffer B. The purity of MAP was confirmed by SDS-polyacrylamide gel electrophoresis using the Laemmli system (18), as shown in Fig. 3.

Protein Sequencing and Activity Measurements-The NH*- terminal sequence of MAP was analyzed by Applied Biosys- terns 477A and 120A systems using automatic Edman degra- dation (19). The inhibition of in vitro protein synthesis was

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Expression and Secretion of MAP by E. coli

Time (hr.)

FIG. 3. Course of MAP production over time. Closed circles, open circles, and triangles indicate the production of MAP in cytosol, culture medium, and periplasmic space, respectively.

determined using rabbit reticulocyte (20) and wheat germ (21, 22) systems with tobacco mosaic virus RNA as mRNA. The inhibitory effect was also examined in an E. coli system with MS2 phage RNA. The protein synthesis system of E. coli was prepared according to Nirenberg and Matthaei (23). MS2 phage RNA was obtained as described by Shimura et al. (24).

RESULTS AND DISCUSSION

Construction of the Secretion Vector of MAP-MAP was found to inhibit the in vitro protein synthesis of an E. coli system and to affect the growth rate of the host strains described below. This seemed to be the major reason for the low production of MAP in E. coli reported previously (2). In order to improve the yield of MAP, a new expression vector, pSH7, was constructed based on the following considerations. First, the expression of the gene should be suppressed until the host cells grow sufficiently, and, second, the gene product should be secreted promptly from the cytosol of the host.

The plasmid pSH7 was constructed to express MAP gene under strict temperature control with PL promoter and ~1857 repressor in a lysogenic h phage system of E. coli. Since PL promoter is strongly repressed by ~I857 repressor, a temper- ature-sensitive mutant of c1 repressor, the plasmid closely regulated the expression of MAP gene placed downstream from the promoter. The PL promoter turns on when ~I857 repressor is inactivated by elevating the culture temperature from 30 to 42 “C at the log-phase of cell growth.

For E. coli to secrete a protein, a signal peptide sequence is necessary at the NH, terminus of the mature protein. OmpA protein is a major outer membrane protein that is produced in large quantities and secreted by E. eoli. Thus, the ompA signal sequence was placed upstream from the MAP gene to enable secretion of the product.

pSH7 is a high copy number plasmid carrying an origin of pUC19. The Shine-Dalgarno sequence of &galactosidase gene, which is expressed as a major protein in E. coli, was introduced downstream from the PL promoter. Furthermore, pSH7 carried a JVdeI site (CATATG) containing the methio- nine codon for initiation of translation to facilitate the inser- tion of a foreign gene.

Expression of MAP Gene and Secretion of MAP-Fig. 3 shows the course of MAP production over time by the E. coli transformant MM294/pSH7. The quantity of MAP in the cytosol, the periplasmic space, and the culture medium were determined by enzyme-linked immunosorbent assay (2). Be- fore induction by the temperature increase, MAP was not detected. One hour after induction, MAP was detected in the cytosol and the culture medium at concentrations of 91 and 25 Kg/liter, respectively. The concentration of MAP in the cytosol and the medium changed as follows: 54 and 116 pg/ liter (2 h), 11 and 143 pg/liter (3 h), and 6 and 140 pg/liter (4 h). MAP was not detected in the periplasmic space at all, as

shown in Fig. 3. The sum of the amount of MAP in the cytosol and that in the culture medium was constant starting 2 h after induction. The amount of MAP in the medium was also constant at a concentration of 140 rg/liter starting 3 h after induction. MAP production began within 2 h after induction and it was gradually secreted into the medium. Most of the product was secreted into the culture medium within 3 h. The transformant grew logarithmically just after induction but stopped growing within only 1 h. The product still seemed to interfere with the growth of the transformant. However, the expression system using the plasmid pSH7 produced enough to be useful for the further study of MAP.

Before the construction of pSH7, two types of vectors were constructed for the expression of the MAP gene. One was pSH6, which contains of ~I857 repressor, PL promoter, and the MAP gene without the ompA signal sequence. E. coli transformant (MM294/pSH6) produced 20 pg of MAP in 1 liter of culture in the first hour after induction, but it did not produce any more thereafter. The other vector was pMH3, which contains lucF repressor and tuc promoter. However, the introduction of the MAP gene was a failure with pMH3. These results indicate that ~I857 may regulate of PL promoter more closely than 1ucP regulates tuc promoter. The transform- ants of pSH7 and pSH6 showed the same growth rate as that of pSH5, which lacked both the ompA signal sequence and the MAP gene before induction by the temperature increase. After induction, the growth of both pSH7 and pSH6 transfom- ants stopped within 1 h, whereas that of pSH5 continued. It is clear that the products of the MAP expression vector influenced the host. The difference in MAP production by the transformants of pSH6 and pSH7 arises from the ompA signal sequence. ompA-linked MAP might be less toxic than native MAP in the cytosol.

It should be noted that MAP was secreted into the culture medium. The secretion of recombinant gene products into the culture medium has been reported, e.g. IgA protease (25), alkaline phosphatase (26), and penicillinase (27). The first report (25) proposed that the protein was secreted through the outer membrane with the processing of an extra domain at the carboxyl terminus. The second (26) showed that the protein was secreted into the medium using host strains, such as a “leaky” mutant of E. coli. In this case, the secretion involved a change in the composition of the outer membrane proteins. The last (27) showed that the protein was secreted into the medium due to the induction of kil gene, which made the outer membrane more permeable. The system used here did not seem to involve those extra domains, the leaky E. coli and the kil gene. Recently it has been reported that ompA- fused ricin B-chain gene can be expressed in E. coli and that the product remains in the periplasmic space (28). So far, there has been no report that the ompA signal sequence causes the secretion of a fused protein into the culture medium.

The temperature increase to 42 “C to induce protein syn- thesis might cause an increase in the permeability of the outer membrane and the subsequent secretion into the culture medium. To confirm the change in permeability, the activity of @-lactamase, a secretion protein whose gene was coded in the plasmid pSH7 (Fig. l), was measured. The activity was 0.3 unit/ml in the cytosol, 2.5 units/ml in the periplasmic space, and 0.3 unit/ml in the culture medium before the temperature increase. One hour after the temperature in- crease, the activities in cytosol, periplasm, and medium were 0.2, 2.6, and 0.8 units/ml, respectively, and they changed as follows: 0.1, 1.3, and 2.1 (2 h), 0.1, 0.7 and 2.5 (3 h), and 0.1, 0.5, and 2.8 (4 h). After the temperature increase, the activity in the medium gradually increased whereas that in periplasm

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decreased. The sum of the activities in the medium and the periplasm was constant. In the medium, the increase in p- lactamase activity corresponded to the increase in the amount of MAP. So, the secretion could be caused by the increase in the permeability of the outer membrane.

Purification of Secreted MAP-MAP was precipitated from the culture medium of E. coli transformant (MM294/pSH7) with ammonium sulfate with a recovery of 50-70%. MAP from the precipitate was purified to homogeneity in two simple steps. The precipitate was dissolved in Buffer A (10 mM sodium phosphate, pH 6), dialyzed against Buffer A, and applied to a column of carboxymethyl-Sepharose equilibrated in Buffer A. MAP was eluted with NaCl in Buffer A at a concentration of approximately 180 mM. The fractions were dialyzed against Buffer B (10 mM Tris-HCl, pH 8.0), and applied to a column of Blue-Sepharose equilibrated in Buffer B. MAP was eluted with NaCl in Buffer B at a concentration of approximately 50 mM. The recovery from the two column chromatographies was approximately 100%. The peak frac- tions were dialyzed against deionized water. At this step, MAP was free from other proteins, and SDS-polyacrylamide gel electrophoresis showed a single protein band (Fig. 4).

The MAP purified from the medium showed the same mobility as native MAP from the Mirabilis plant in SDS- polyacrylamide gel electrophoresis (Fig. 4). The first 15 NHZ- terminal amino acid residues of the secreted MAP were de- termined to be Ala-Pro-Thr-Leu-Glu-Thr-Ile-Ala-Ser-Leu- Asp-Leu-Asn-Asn-Pro, and they coincided with those of na- tive MAP. This result shows that the cleavage occurred pre- cisely between the ompA signal and MAP.

Inhibition of Protein Synthesis by MAP-The inhibitory effect of both the secreted and native MAP was measured on the in uitro protein synthesis of rabbit reticulocyte, wheat germ, and E. coli systems. The results were compared with those of ricin A-chain (Figs. 5, a, b, and c). Both MAPS showed an IC&, of 2 nM for rabbit reticulocyte (Fig. 5a), 3 nM

for wheat germ (Fig. 56), and 200 nM for E. coli (Fig. 5~). Complete inhibition by both the secreted and native MAP was observed at 10 nM for wheat and rabbit, and at 1 pM for E. coli (Figs. 5, a, b, and c). Thus the inhibitory effect of the secreted MAP was the same as that of native MAP. Ricin A- chain had an I& of 0.067 nM for rabbit reticulocyte (Fig. 5a). It was approximately one-thirtieth that of MAP. On the other hand, MAP completely inhibited the protein synthesis of wheat germ at a concentration of 10 nM, whereas ricin A- chain did not show any effect at the same concentration. Even 100 nM of ricin A-chain showed only 35% inhibition in the wheat germ system (Fig. 5b). No effect of ricin A-chain on the E. coli system was observed even at 2 pM, whereas com-

1 2

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29’ w-

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FIG. 4. SDS-polyacrylamide gel electrophoresis of purified MAP. Lane I indicates native MAP from the Mirabilis plant, and lane 2 indicates the MAP secreted by E. coli.

FIN. 5. Inhibitory effect of the secreted and native MAP, and ricin A-chain on in vitro protein synthesis in rabbit reticulocyte (a), wheat germ (b), and E. coli (c) system. Open circles, closed circles, and triangles indicate the MAP secreted by E. coli, native MAP from the Mirabilis plant, and ricin A-chain, respec- tively. The relative activities are presented in terms of the incorpo- ration of L-[““Slmethionine without mRNA as a zero percent control.

plete inhibition by MAP was observed at 1 pM (Fig. 5~). MAP inhibited the protein synthesis of the E. coli system substan- tially, while ricin A-chain did not show any effect at all. This could be the main reason for the low expression of MAP as an intracellular protein.

The inhibitory effect of MAP on the protein synthesis of rabbit reticulocyte and wheat germ was different from that of ricin A-chain. Ricin A-chain inactivated mammalian ribo- somes rather than those from plants (3). The difference between MAP and ricin A-chain might be due to their selec- tive effect on those ribosomes.

Surprisingly, MAP inhibited the protein synthesis of E. coli, whereas ricin A-chain did not show any effect at all. E. coli ribosomes have been considered to be essentially insen- sitive to the action of RIPS. RIP cleaves an N-glycosidic bond at A4”“” in rat liver 28 S rRNA, and it has been suggested that the cleavage inactivates the ribosome (9). A’,‘““ is located in a region that is highly conserved in the rRNA (29). This region is also conserved in E. coli 23 S rRNA (29); A”“““corresponds to A“‘“” in rat liver 28 S rRNA. It has been suggested that the A”““O in E. coli rRNA interacts directly with the E. coli elon- gation factor EF-G and EF-Tu (30). The cleavage of the N- glycosidic bond might involve the loss of elongation factor- dependent functions in the ribosomes. The N-glycosidic bond at A’“‘” in E. coli 23 S rRNA might be cleaved by MAP and this might result in the inactivation of the ribosome.

Although an intact ribosome of E. coli is resistant to ricin, the adenine base of the deproteinized E. coli 23 S rRNA is released by a high concentration of ricin A-chain (31). This suggests that r-protein(s) might act to protect the rRNA from

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10992 Expression and Secretion of MAP by E. coli

ricin attack. MAP inhibited the protein synthesis of E. coli but ricin A-chain did not. This might arise from the different recognition of the r-protein(s).

There are meaningful conservations in the amino acid sequence of MAP and ricin A-chain. However, their effects are quite different with regard to the inactivation of the protein synthesis of E. co&. The difference might be caused by the different sequences in MAP and ricin A-chain. The MAP secretion system using the plasmid pSH7 facilitates the production of MAP mutants. This is useful not only in the study of the mechanism by which protein synthesis is inhib- ited but also in the study of specificity, that is, how particular ribosomes are recognized.

Acknowledgments-We wish to thank Dr. Yaeta Endo, Yamanashi Medical College, for his kind advices in the evaluation of E. coli protein synthesis, Miho Ohkubo for the synthesis of oligodeoxynucle- otides, Dr. Yoshisuke Nishi for his critical reading of this manuscript.

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Expression and secretion of Mirabilis antiviral protein in Escherichia coli and its

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