Plant Cell Reports

Plant Cell Reports (1996) 15:910-913

9 Springer-Verlag 1996

Production of ribosome-inactivating protein from hairy root cultures of Luffa cylindrica (L.) Roem. Luigi Sanitd di Toppi i, Paola Gorini 2, Giuliana Properzi I, Luigi Barbieri 2, and Laura Span61 1 Dipartimento di Biologia di Base ed Applicata, Universitd degli Studi di L'Aquila, 1-67100 Coppito L'Aqnila, Italy 2 Dipartimento di Patologia Sperimentale, Universit& degli Studi di Bologna, via S. Giacomo 14, 1-40126 Bologna, Italy Received 1 June 1995/Revised version received 10 January 1996 - Communicated by M. R. Davey

ABSTRACT Transformed root lines of Luffa cylindrica (L.) Roem. (Cucurbitaceae) were established by inoculation of in vitro grown plantlets with wild type Agrobacterium rhizogenes strain 1855. Cloned lines of hairy roots were tested for the presence of ribosome-inactivating proteins; crude extracts inhibited protein synthesis in a reaction mixture based on rabbit reticulocyte lysate. Inhibitory activity increased during culture period, reaching a maximum value in the stationary phase. No activity could be detected in the culture medium, nor in extracts from callus and/or suspension cultures. A ribosome-inactivating protein having specific activity of 62,100 U mg protein -1 and a molecular mass of 26-28,000 Da was purified to homogeneity. The protein showed N-glycosidase activity on rat liver ribosomes. The results demonstrate that hairy root cultures can be successfully utilized for the in vitro production of ribosome-inactivating proteins. Abbreviations: BAP:benzylaminopu rine;2,4D:2,4dichlorophenoxyacetic acid; HPLC: high pressure liquid chromatography; MS: Murashige and Skoog; NAA: naphthaleneacetic acid; NCPPB: National Collection of Phytopathogenic Bacteria; Ri: root-inducing; RIP : ribosome-inactivating protein; UV: ultra -violet; YMB: yeast mannitol broth. INTRODUCTION Ribosome-inactivating proteins, RIPs, are enzymes of plant origin which deadenylate ribosomal RNA at a specific site, corresponding to A4324 in 28S rat rRNA (for recent review see Barbieri et al. 1993). During the past ten years an increasing body of information has been produced by different research groups, on the properties and biological function of these proteins. In particular, attention of most investigators has been devoted to the exploitation of their potential use i) in agriculture, as antiviral and/or antifungal factors to be utilized for plant protection; ii) in medicine, as Correspondence to: L. Span6

components of cytotoxic conjugates with specific monoclonal antibodies (immunotoxins). In this context, one of the major drawbacks to face during the purification of RIPs with high purity grade and low pyrogenic activity (ability to elicit fever in higher vertebrates), is that very often the starting material is heavily contaminated with environmental detritus and bacteria. Cultures of plant tissues and/or cells, grown in vitro under sterile conditions, may overcome this problem, by constituting a pyrogen-free starting material ideally suited for the preparation of products to be used in experimental clinical medicine. However, several reports (Misawa et al. 1975; Ikeda et al. 1987; Bonness and Mabry 1992; Thomson et al. 1991; Barbieri et al. 1989) concerned with traditional tissue culture systems have shown that in vitro cultures are frequently unstable and very variable in their capacity to guarantee a high yield of RIPs. Transformation of plants by infection with Agrobacterium rhizogenes results in the transfer of part of the Ri (root-inducing) piasmid and in the proliferation of adventitious roots ("hairy roots") generally localized at the site of infection (Chilton et al. 1982; Span6 et al. 1982). Hairy root cultures offer an intrinsic higher stability (Flores 1987; Aird et al. 1988; Inomata et al. 1993), grow rapidly in vitro (Tepfer 1990), and can be engineered with foreign genes (Comai et al. 1985; Hamill et al. 1987; Firoozabady et al. 1994), thus being a better candidate for an in vitro RIP-producing system. Furthermore, the possibility of establishing and optimizing the growth of hairy root cultures in laboratory scale pilot plant bioreactors seems to be a realistic prospect (Rhodes et al. 1986; Hilton et al. 1988; Taya et al. 1989). Recent encouraging results have been reported by Savary and FIores (1994) for hairy root cultures of Thricosanthes kirilowii. The research presented in this paper was undertaken to verify the possibility of obtaining hairy root lines capable of stably producing RIPs in

911 amounts suitable for mass production. RIPs from Luffa cylindrica (L.) Roem. (luffins) are already known (Kishida et al. 1983; Kamenosono et al. 1988; Watanabe et al. 1989; Islam et al. 1990; Islam et al. 1991 a; b; Ng et al. 1992) and are good candidates for all the uses proposed for RIPs. MATERIALS

AND

Table 1. Distribution of RIP activity Translational inhibitory activity in extracts from Luffa cylindrica (L.) Roem. tissues. One unit is defined as the amount of protein necessary to inhibit protein synthesis by 50% in the rabbit reticulocyte lysate system. ............................................................................

Tissue

Specific activity (U mg protein -1)

METHODS:

Total activity (U g tissue"1)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Plant material and bacterial strains:

Agropine-type Agrobacterium rhizogenes strain NCPPB 1855 was maintained on YMB medium (Hooykaas et al. 1977). Seeds of Luffa cylindrica (L.) Roem. were surface sterilized by immersion in 70% (v/v) ethanol for 15 min., followed by 20 min incubation in 20% NaCIO and rinsing several times in sterile distilled water, Seeds were then germinated on Gambcrg's B5 basal medium (Gamborg et al. 1968) and utilized for infection with Agrobacterium rhizogenes after one month. Stems of in vitro grown plantlets were directly infected by pinching with a scalpel previously dipped in a single colony isolate from solid cultures grown at 28 ~ C for 48 h. Transformed roots emerged in five or six weeks. They were explanted and transferred to hormone-free B5 medium supplemented with 250 mg ml-1 of cefotaxime. Transformation was checked by Southern hybridization (Southern 1975), utilizing EcoRI fragment 15 as probe. Root tips of ca. 2.5 cm in length were cut and transferred into 250-ml flasks, containing 50 ml of hormone-free B-5 liquid medium. Root cultures were grown in the light (ca. 65 mE m-2s -1) at 25~ on a rotary shaker (100 rpm) and subcultured every 30 days. Samples were randomly harvested at 5-day intervals, dried on tissue paper and a fresh weight growth curve generated. A culture of normal (not transformed) roots was established as a control. Explants from young leaves, stems and roots of in vitro grown plantlets of Luffa cylindrica (L.) Roem. were placed on B5 solid medium supplemented with 1 mg I-1 2,4-D; 1 mg 1-1 NAA and 0.5 mg I1 BAP. In some experiments kinetin was substituted for BAP without modifying the results; callus cultures were maintained on this medium in a growth chamber (18 h illumination period, 25~ and subcultured every 25 days. Cell suspensions were prepared by inoculating about 1.5 g of callus in 250-ml flasks containing 50 ml of B5 liquid medium supplemented with 1 mg 1-1 NAA and 0.5 mg 1-1 BAP. After a few days of acclimatization cells were filtered on 150 mm mesh filter and suspended at a density of about 70,000 cells ml 1 in fresh liquid medium. Cells viability was evaluated by using fluorescein diacetate. The cell suspension was grown at 25~ in the light (ca. 65 mE m-2sl), on an orbital shaker at 120 rpm and subcultured every 25 days.

Seeds 5,400 Young leaves 400 Old leaves 833 Callus inactive Cell suspension inactive Hairy roots 1,600 -13,000a Normal roots 345

RESULTS AND DISCUSSION: Distribution in the plant: RIP activity of Luffa cy/indrica(L.) Roem. plantlets, grown in vitro on MS medium, was evaluated in crude extracts from different parts and organs and compared to the inhibitory activity shown by extracts from seeds and from transformed roots.The results of such an analysis are reported in Table I.

11,300 -162,600 4,290

a - see results in figure 1 As can be seen the distribution of inhibitory activity, as far as normal, non-transformed tissues are concerned, is in agreement with what was already known from previous reports (Kamenosono et a1.1988; Watanabe et al. 1989). Maximum activity is in fact present in extracts from seeds and is drastically reduced (by a factor of ten) in extracts from roots and leaves. In this latter case, however, we have found that old leaves reproducibly showed higher activity than young ones; also this result is in good agreement with recent findings on the inhibitory activity of senescent tissues from other RIP-producing plants (Barbieri et al. in preparation).

RIP purification:

Tissues were frozen in liquid nitrogen and homogenized in the ratio of 1:4 (w/v) in phosphate buffered saline (PBS= 0.14 M NaCI in 5 mM Na phosphate buffer, pH 7.5). Seeds were washed under tap water and ground for 5 min with an Ultra-turrax homogenizer in cold PBS. Each homogenate was stirred overnight at 4~ filtered through gauze and centrifuged at 10,000 g for 25 min. Protein content was determined spectrophotometrically (Kalb and Bernlhor 1977). Inhibition of protein synthesis (RIP activity) of crude extracts and culture media was measured at 28~ in a reaction mixture based on rabbit reticulocyte lysate (Ferreras et al. 1993). Activity was expressed as specific t activity (units mg protein -1) or total act'vity (units g tissue- 1). One unit corresponds to the amount of protein that inhibits by 50% the protein synthesis in the rabbit reticulecyte lysate. Crude extracts was applied to an S-Sepharose column (13x2 cm) equilibrated in 20 mM Na acetate, pH 4.5, at room temperature. The column was washed with 5 mM Na-phosphate buffer, pH 7.0 and the bound protein eluted with O.5M NaCI in 20 mM Na phosphate buffer pH 7.0. The S-Sepharese fraction was subjected to gel filtration on Sephadex G-25 equilibrated and eluted in 5 mM Na phosphate buffer pH 7.0. Excluded proteins were applied to a second S-Sepharose column (18xl cm) and eluted with a linear gradient (0-0.5 M NaCI) at a flow rate of 50 ml h 1 . Active fractions were pooled, dialyzed against distilled water and subjected to micropreparative gel filtration on a column of G3000SW (Toso-Haas, Stuttgard) equilibrated and eluted in 0.1 M Na phosphate buffer pH 6.7, containing 0.1 M Na2SO 4. Direct enzymatic determination of RIP activity was by adenine release from rat liver ribosomes (Barbieri et al. 1992). Molecular mass was evaluated by HPLC gel filtration.

73,000 26,700 20,200

12

15

~i!ilijii! 10 ~ r-

r--

u.

5

0

f

0

5

10

15

20

~

25

30

Culture time (days) " FIGURE 1. Growth curve and specific translational inhibitory acttvity of Luffa cylindrica (L.) Roem. hairy roots crude extracts. Bars, fresh weight; line, specific activity.

Callus and cell suspension cultures: Callus and cell suspension cultures were obtained from both normal and transformed tissues of Luffa cylindrica (L.) Roem. They showed maximal growth

912 Table 2 . Luffins purification from Hairy roots of Luffa cylindHca (L.) Roem. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Purification step

Protein mg

Specific activity (U mg protein"1)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Crude Crude acidified S-Sepharose I G-25 S-Sepharose II Gel filtration

-t ..............

280.00 234.00 26.30 3.52 0.46 analytical

Total activity (U g tissue-1)

Yield %

%. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

796 931 1,923 13,870 38,911 62,100

when the medium was supplemented with 30 g L -1 sucrose, lmg L -1 NAA and 0.5 mg I"1 BAP. Other hormone concentrations had detrimental effects on cell growth and viability. In all cases, no translational inhibitory activity was detectable either in extracts from plant material or in the culture medium (not shown). Hairy root cultures: About 80% of the infected plantlets of Luffa cylindrica (L.) Roem. formed vigorous hairy roots within four weeks from the inoculation of the bacteria. Excised roots were grown successfully in tissue culture; optimal growth conditions included light and the omission of phytohormones from Gamborg's B5 medium (see experimental procedure). Under these conditions, hairy root cultures showed a sigmoidal growth curve and crude extracts a progressively increasing translational inhibitory activity, that reached the maximum value during the early stationary phase (see fig.l). No detectable translational inhibitory activity was ever observed in the hairy root culture medium (not shown).

222,320 217,854 50,577 48,821 17,899 analytical

100,0 98,0 22,7 22,0 8,1 analytical

Purification of ribosome-inactivating protein from hairy roots: The purification scheme which usually gives rise to pure RIP starting from other plant tissues does not give pure protein after the step of the second S-Sepharose column in the case of Luffa cy/indrica (L.) Roem. (see Table 2 and Figure 2).Contaminants seem not to be proteinaceous, as deduced from the UV spectrum. Pure protein could be obtained by micropreparative gel filtration (Figure 3); the UV spectrum of this preparation is similar to that obtained for other RIPs. The protein was purified to homogeneity by further analytical gel filtration under the same conditions. The molecular mass, calculated by retention time, is in agreement with that reported for known luffins: 26-28,000 daltons. 100

0.075 26-28 kDa

0.05

g

g

r

- 100

0.08

~" 0.025 9

- 8 0 =

I

0.4

0.06

E 9

Z

g

(3 m

"~ 0.o4

1

0

tO

: -40 J

o

4 0.2"

10

20

Retention time (rain}

< 0.02 211 ~.

i

I 0

i

I

i

20

4o

60

J

80

0

~

100

Fraction no. FIGURE 2. Elution pofile and translational inhibitory activity of

from the second cation exchange chromatograpy ~Sroteins Sepharose II) of extracts from hairy roots of Luffa cylindrica (L.)

Roem. Continuous thick line, A280; dotted line, inhibition of translation; thin line, NaCI gradient.

FIGURE 3. HPLC gel-filtration of the ribosome-inactivating proteins purified from hairy roots of Luffa cylindrica (L.) Roem. Bars, cell-free translation; line, A280

The purified protein was tested for N-glycosidase activity on rat liver ribosomes: 0.74 mol of adenine per mol of ribosomes were released in 10 minutes at 37~ at a RIP/ribosome ratio of 1:10. The results reported in the present paper demonstrate that RIPs can be produced and purified from hairy root cultures, in good agreement with what has been recently reported by Savary and

913 Flores (1994) for hairy root lines of Thricosanthes kirilowii. The translational inhibitory activity found in extracts from our hairy root cultures is the highest that has been found in various tissues of Luffa cylindrica, including seeds. The identity of the form of luffins produced by hairy roots has not been yet clarified and could be different from the naturally occurring RIPs of Luffa cylindrica (L.) Roem. seeds. However, this would not constitute a real drawback for its utilization, since there are no indications that a particular isoform may be a better choice for exploitation in immunotoxin preparation or in agriculture. The possibility of producing RIPs in a nonbacterial system is very promising. In fact, systems based on the expression of recombinant proteins in Escherichia coil have the major drawback of RIP toxicity to bacterial ribosomes, since they are free in the cytoplasm, and nascent proteins do not segregate in a membranous compartment. Using an autologous system to produce RIPs should be much safer for the protein synthesis machinery, thus allowing a higher accumulation of final product, without killing the host cell. Signal peptides for the export of plant proteins are already known and they may be engineered in a recombinant RIP product which may then be secreted in the medium, thus simplifying the process of RIP recovery. CONCLUSION RIP-producing hairy roots promise to be much more stable than conventional in vitro grown calluses and cell suspensions. Furthermore they appear to be a very convenient system for the production of proteins to be used in pharmaceutical industry since they may be both sterile and pyrogen-free. ACKNOWLEDGMENTS: This work was partially supported by C.N.R., Progetto Finalizzato Biotecnologie e Biostrumentazione, By AIRC and by Pallotti's Legacy for Cancer Research. REFERENCES: Aird ELH, Hamill JD, Robins RJ, Rhodes MJC(1988) In: Robin RJ, Rhodes MJC (eds) Manipulating secondary metabolism in culture. Cambridge University Press, Cambridge, pp137-144 Barbieri L, Battelli MG, Stirpe F (1993) Biochim Biophys Acta 1174 : 237-282. Barbieri L, Bolognesi A, Cenini P, Falasca AI, Minghetti A, Garofano L, Guicciardi A, Lappi D, Miller SP, Stirpe F (1989) Biochem J 257: 801-807. Barbieri L, Ferreras JM, Barraco A, Ricci P, Stirpe F (1992) Biochem J 274:1-4 Bonness MS, Mabry TJ (1992) Plant Cell Reports 11 : 6670 Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart F Temp6 J (1982) Nature 295: 432-434. Comai L, Facciotti D, Hiatt WR, Thomas G, Rose RE Stalker DM (1985) Nature 317:741-744

Ferreras JM, Barbieri L, Girb6s T, Battelli MG, Rojo MA, Arias FJ, Rocher MA, Soriano F, Mendez E, Stirpe F (1993) Biochim Biophys Acta 1216:31-42 Flores HE (1987) In: Mumma RO, Lebaron H, Honeycutt RC, Duesing JH (eds) Applications of biotechnology to agricultural chemistry. Am Chem Soc Symp Series 334, Washington DC, pp 67-86 Firoozabady E, Moy Y, Courtney-Gutterson N, Robinson K (1994) Bio/Technology 12: 609-613. Gamborg OL, Miller RA, Ojima K (1968) Exp Cell Res 50 : 151-158 Hamill JD, Prescott A, Martin C (1987) Plant Mol.Biol 9 : 573-584 Hilton MG, Wilson PDG, Robins RJ, Rhodes MJC (1988) In: Robin RJ, Rhodes MJC (eds) Manipulating secondary metabolism in culture. Cambridge University Press, Cambridge Hooykaas PJJ, Klapwjik P, Nuti MP, Schiiperoort RA, Rorsch A (1977) J Gen Microbiol 98 : 477-484 Ikeda T,Takanami Y, Imaizumi S, Matsumoto T, Mikami Y, Kubo S (1987) Plant Cell Reports 6 : 216-218 Inomata I, Yokoyama M, Gozu Y, Shimizu T,Yanagi M (1993) Plant Cell Reports 12:681-686 Islam MR, Nishida H, Funatsu G (1990) Agric Biol Chem 54:1343-1345 Islam MR, Hirayama H, Funatsu G (1991)a Agric Biol Chem 55 : 229-238 Islam MR, Kung SS, Kimura Y, Funatsu G, (1991)b Agric Biol Chem 55 : 1375-1381 Kalb VF ,Bernlohr RW (1977) Anal Biochem 82 : 362-371 Kamenosono N, Nishida H, Funatsu G (1988) Agric Biol Chem 52:1223-1227 Kishida K, Masuho Y, HaraT (1983) FEBS Lett 153 : 209-212 Misawa M, Hayashi M, Tanaka H (1975) Biotech and Bioeng 17:1335-1347 Ng TB, Wong RNS, Yeung HW (1992) Biochem Int 27: 197-207. Rhodes MJC, Hilton M, Parr AJ, Hamill JD, Robins RJ (1986) Biotech Lett 8 : 425-420 Savary BJ, Flores HE (1994) Plant Physio1106 : 11951204. Southern EM (1975) J Mol Biol 98 : 503-517 Span5 L, Pomponi M, Costantino P, Van Slogteren GMS, Temp6 J (1982) Plant Mol Biol 1 : 291-300 Taya M, Yoyama A, Kondo O, Kobayashi T, Matsui C (1989) J Chem Engin Japan 22 : 84-89 Tepfer DA (1990) Physiol plantarum 79 : 140-146 Thomsen S, Hansen HS, Nyman U (1991) Planta Medica 57 : 232-236 Watanabe K, Suemasu Y, Funatsu G (1989) J Biochem 106 : 977-981