Biotechnol. Bog. f994, 10, 345-352
ARTICLES Ribosome-Inactivating Protein Production from Trichosanthes kirilo wii Plant Cell Cultures John E. ThorupJ Karen A. McDonald,'p? Alan P. Jackman,?Nishant Bhatia,f and Abhaya M. Dandekad Departments of Chemical Engineering and Pomology, University of California, Davis, California 95616
Two ribosome-inactivating proteins (RIPs) found in Trichosanthes kirilowii root tuber, trichosanthin (27 kDa) and TAP-29 (29 kDa), have been reported to exhibit antiviral (including HIV-1) and antitumor activities. Using SDS-PAGE and Western blotting analyses, we have studied the production of intracellular and extracellular proteins from T. kirilowii callus grown on semisolid medium, callus grown in suspension, and Agrobacterium rhizogenes A4 transformed callus grown in suspension. This transformation resulted in callus rather than hairy root growth, although the growth rate of the transformed callus on hormone-free medium was similar to that obtained for the nontransformed callus on hormone medium. Trichosanthin, identified through SDSI PAGE and Western blotting, was detected only in root tuber and cell extracts of the transformed cell line. A 29-kDa protein was found in intracellular extracts and extracellular solutions from all of the above samples; however, the highest yield was obtained from the broth of the agrobacterium-transformed callus. Following ionexchange purification of the shake flask broth on a strong cation-exchange column (S-Sepharose), elution fractions containing the 29-kDa protein showed a high degree of RIP activity, as evidenced by total inhibition of protein synthesis using an in vitro protein translation assay. The yield of the 29-kDa protein recovered from the broth was greater than 1.0% (w/w) of the dry weight of the callus. For comparison, the yield of TAP-29 obtained by extraction of dried root tuber is on the order of 0.01% (w/w) of the dry weight (Lee-Huang et al., 1991);our estimate of the 29-kDa protein from our fresh root tuber is between 0.4% and 1%(w/w) on a dry weight basis.
Introduction Plant cell cultures may be an attractive approach for obtaining high-value plant proteins, particularly those which undergo processing (e.g., glycosylation),modification, or assembly in vivo and/or are difficult to express in recombinant microbial systems. Ribosome-inactivating proteins (RIPS) are plant proteins named for their damaging effect on eukaryotic ribosomes [see the recent review article by Stirpe et al. (199211. They are present inunrelated families of the plant kingdom and are possibly ubiquitous in nature (Stirpe and Barbieri, 1986; Barbieri et al., 1989). Two classes of RIPS have been identified. Type I RIPS are single-chain proteins, sometimes glycosylated, ranging in molecular mass from 23 to 32 kDa with a PIof 8-10. Type I1 RIPs, composed of two chains, the A and B chains,joined by one or more disulfide bonds, are typically 60-65 kDa, glycosylated,with a PIranging from 4.8 to 8.0. Although the biological function of RIPS in nature is unknown, their mechanism of activity against eukaryotic and, more recently, prokaryotic ribosomes is well-characterized (Endo et al., 1987, 1988; Endo and Tsurgi, 1987, 1988; Hartley et al., 1991). Although RIPS have interesting pharmacological prop-
* Author to whom correspondence should be addressed. + Department of Chemical Engineering. t Department of Pomology.
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erties, there have been very few reports of plant cell and/ or tissue culture studies either on-semisoGd media or in liquid suspension. Barbieri et al. (1989) initiated plant callus cultures from the leaf tissue of four RIP-producing plants-Phytolacca americana (pokeweed), Saponaria officinalis (soapwort),Dianthus caryophyllus (carnation), and Zea mays (corn)-and demonstrated the importance of the type and concentration of synthetic hormones in the media on RIP production. Another interesting observation was that the RIP-producing pokeweed callus line contained three distinctly different proteins, which all showed inhibition of protein synthesis. Ikeda et al. (1987a-q 1988a,b)studied the production of MAP (Mirabilis antiviral protein), a type I RIP, fromMirabilis jalapa callus cultures. They also observed a strong influence of synthetic hormones on MAP production, with increasing levels of synthetic hormones suppressingboth growth rate and MAP production. Thomsen et tal. (1991)studied the production of RIPS from Phytolacca dodecandra. One of the most interesting observations of their study was that the callus produced two new RIPS with slightly higher molecular weights that were not previously found in intact plant tissue. In contrast,the well-known RIP dodecandrin, found in leaf tissue, was not found in their cultures. Bonness and Mabry (1992) also studied RIP production in Phytolacca dodecandra callus and suspension culture, noting the variability of RIP production from different
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callus cell lines, the absence of dodecandrin in most of the cultures, and the presence of other immunoreactive RIPS in callus and suspension cells. While most of the literature has focused on RIPSfound intracellularly, very few studies have characterized extracellular proteins or RIP activity from the broth of plant cell suspension cultures. In our study, we focus on ribosome-inactivating proteins from the Cucurbitaceae family, which possesses a variety of potentially useful biological activities such as antiviral antitumor, antidiabetic, abortifacient, and immunomodulatory [see the review by Ng et al. (1992)l. Of particular interest are two RIPS found in Trichosanthes kirilowii, trichosanthin and TAP-29, which have been identified as anti-HIV compounds (McGrath et al., 1989; Lee-Huang et al., 1991). Trichosanthin, a 27-kDa protein found in root tubers of T. kirilowii, is currently undergoing phase I1 clinical trials as an AIDS drug (Byers et al., 1990;Kahn et al., 1990). Initial reports of in vitro studies indicate that TAP-29's anti-HIV activity is as potent as trichosanthin's but it is less toxic to intact, healthy cells (LeeHuang et al., 1991). Previous literature indicates the importance of hormone levels on growth and RIP production in plant cell culture, and it is well-known that transformation of plant cells by infection of Agrobacterium rhizogenes can alter intracellular levels of auxins and cytokinins (White et al., 1985; Estruch et al., 1985; Estruch et al., 1991a,b). Transformation of plant cells by infection with Agrobacterium rhizogenes can result in the transfer of DNA from the bacterium to the plant. These bacteria harbor the rootinducing Ri plasmid causing the proliferation of roots at the site of infection (Moore et al., 1979;White and Nester, 1980a,b;Gheysenet al., 1985). The transfer andexpression of a segment of the Ri plasmid, the Ri T-DNA, in the plant cells result in the formation of "hairy roots" (Chilton etal., 1982;Spanoetal., 1982;Whiteetal., 1982;Willmitzer et al., 1982) and are sometimes accompanied by the expression of opines that are not normally produced by the plant cells. However, opines are not always expressed by the transformed tissue (Flores et al., 1987; Uratsu et al., 19911, and in some cases callus, rather than hairy roots, has been obtained from infection with A. rhizogenes (White et al., 1982). Hairy root cultures are generally fast-growing, easy to manipulate, and genetically stable (Tepfer, 1989). Hairy roots have also been found to grow well without an exogenoussupply of hormones, which is necessary for callus cultures. For these reasons, there has been considerable interest in the use of hairy root cultures to produce plant products. Savary and Flores (1991) have reported that hairy root cultures of T. kirilowii produce trichosanthin. There have been fewer reports of transformed callus and limited characterization of these cell lines. However, they may possess many of the beneficial features of hairy root cell lines (eg., high growth rates, genetic stability, and elimination of the need for exogeneouslysuplied hormones) with the added advantage of a growth morphology that is easier to culture in a large-scale bioreactor, in terms of inoculation, mixing, aeration, sampling, immobilization, and modeling. In this article, we present our results on the transformation of T. kirilowii with A. rhizogenes and analyses and partial characterization of intracellular and extracellular proteins obtained from the transformed plant suspension cultures. For comparison purposes, we have also analyzed intracellular proteins obtained from T. kirilowii root tuber, nontransformed callus tissues grown
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on semisolid media, and nontransformed callus grown in suspension.
Materials and Methods Media Formulation. The T. kirilowii plant tissue was grown on Linsmaier and Skoog (LS) medium with 3% sucrose at pH 5.7. Stock solutions of the inorganic nutrients (major salts), trace elements (minor salts), organic supplements, and iron sources were prepared according to Dixon (1985). Media with hormones contained 5.0 pM (2,4-dichlorophenoxy)aceticacid (2,4-D) and 1.0 pM (benzy1amino)purine (BAP). Semisolid medium was prepared by adding 9 g/L plant cell culture tested agar prior to autoclaving. Callus was subcultured approximately every 4 weeks onto fresh LS medim. TransformationProtocol. Agrobacterium rhizogenes A4 stored at -80 OC was used to inoculate semisolid plates of modified LB medium. The agrobacteria were incubated at 25 OC and subcultured every 2 weeks. Two techniques were used to inoculate the plant tissue with the A. rhizogenes A4. The first technique involved simply nicking or cutting in half a sterile plantlet at various sites with a scalpel previously dipped into colonies of the agrobacterium. Also, sterile plantlets were wounded by jabbing the site numerous times with a syringe needle dipped in the agrobacterium. The second technique was a modification of a protocol by Dandekar et al. (1989) for the transformation of somatic embryos. This protocol attempted to maximize the virulence of the agrobacterium by optimizing two environmental variables, the pH and the agrobacterial concentration, during inoculation of the plant tissue. Multiple colonies less than 20 days old were used to inoculate 25-mL Erlenmeyer flasks containing 10 mL of modified LB medium. The flasks were put into a constanttemperature orbital shaker at 25 "C and 150 rpm. The bacteria were grown for 10-15 h until the absorbance at 420 nm was no less than 1.6 AU. The cells were spun down at 4000rpm (2400g)for 10min, and the supernatant was discarded. The cells were resuspended by vortexing in 10 mL of fresh, modified LS medium at pH 5.25. The suspension was further diluted with the same medium until the absorbance at 420 nm was 0.5 AU, corresponding to 2.5 X 108 cells/mL of medium. Once the agrobacteria were at the optimum pH (5.25) and concentration (2.5 X lo8cells/mL) for transformation, the plant tissue was inoculated. Under sterile conditions, 1-1.5-cm segments of petiole, stem, leaf, and root tissues were harvested from T. kirilowii sterile plantlets and immersed in the A. rhizogenes A4 suspension. The suspension was briefly swirled and then left undisturbed for 8-10 min. Surface-sterilized, fresh carrot disks were used as controls to monitor the transformation experiment. Uninfected T. kirilowii segments and carrot disks were also used as controls to distinguish effects due to the agrobacterium from wound response, media effects, and/ or antibiotic exposure. In a similar fashion, carrot disks from freshly picked, sterilized carrots were exposed to the agrobacterium suspension as a control. Additional carrot disks were immersed in a solution of sterile, modified LB medium (pH 5.25) as a second control. After the incubation period, the tissue segments and disks were removed and dabbed dry on sterile filter paper. The carrot disks from both controls and the T.kirilowii segments were placed on plates of hormone-free LS semisolid medium. The bacteria and plant tissues were cocultivated for as much as 14 days to investigate the effects of extensive cocultivation on the transformation of plant tissue.
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After the cocultivation period, the T. kirilowii segments were moved to fresh plates of LS hormone-free medium containing cefotaxime. Cefotaxime,an antibiotic, was used to kill the A. rhizogenes A4. A simple toxicity test was performed to determine the most effective level of cefotaxime in the LS hormone-free medium. The test involved placing T. kirilowii and A. rhizogenes A4 on plates containing various concentrations of cefotaxime ranging from 100 to 500 pg/mL. A concentration of 350 pg/mL visually appeared to be the optimal concentration a t which the agrobacteria died and the plant segments were unaffected. The segments remained exposedto cefotaxime for 7-12 weeks and were moved to fresh plates every 7-10 days during this period. Once the agrobacteria had been killed, the plant segments were moved to hormone-free LS medium plates. The transformation produced a tumorous tissue that grows on LS hormone-free medium, which visually appeared be similar to the callus grown on LS medium with hormones. Nearly the same subculturing schedule and procedure were used to maintain the agrobacterium-transformed callus tissue that were used for the conventional callus. The only exception was the use of hormone-free LS medium. Suspension Cultures. Suspension cultures were prepared using callus produced by hormone alteration and by transformation with A. rhizogenes A4. The particular agrobacterium-transformed cell line that was used in the studies was obtained from a transformation experiment that used a 12-day period of cocultivation followed by a 7-week exposure to cefotaxime. Under sterile conditions, calli (from fourth generation material that had been subcultured every 4 weeks on semisolid, hormone-free medium) were sieved through 20-mesh screens by firmly mashing the calli with a small spatula. The fresh weight of the innoculum was measured under sterile conditions. The inoculum was added to 100 mL of the appropriate LS liquid medium (with or without hormones) in a 250-mL Erlenmeyer flask. The shake flasks were maintained in the light at 25 OC and 110 rpm. Two 15-W fluorescent lights were placed 4-6 in. above the orbital shaker and remained on continuously. The results reported are for shake flask cultures harvested after growing for 30 days in suspension. Filtered cells and broths from three separate flasks were combined for the analyses. Protein Extraction and Purification. The separation of the extracellular and intracellular proteins produced in a shake flask culture was accomplished by simply separating the suspension cells from the broth by vacuum filtration using No. 3 Whatman filter paper. Any extracellular protein remaining on the cells was removed by washing the cells on the filter paper with 20 mM PBS buffer (pH 7.0). The washings were then combined with the filtered broth. The volume of the filtrate, as well as the fresh weight of the suspended cells, was recorded. The broth was stored at -20 "C for later analysis. Separation of the proteins produced by the callus grown on semisolid medium was accomplished by gently scraping the clumps of callus off the semisolid medium using a spatula. The fresh weight of the callus and the weight of the semisolid medium were measured. The cells were then gently washed to remove any extracellular proteins by stirring in 20 mM PBS (pH 7.0) to suspend the cells. Then, the cells were gently settled by centrifugation for 10 min a t 2400g. The extracellular proteins in the supernatant were combined with the extracellular protein extracted from the semisolid medium. The extracellular proteins
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in the semisolid medium were extracted using the same protocol used to extract the callus, as will be described here. Extraction of proteins from the cellular material or the semisolid medium was begun by adding 2 mL of 4 OC, 20 mM PBS (pH 7.0) per gram of material to be extracted. The suspension was kept on ice and homogenized using a Tissue Tearer for 30 s followed by 30-s rest, which was repeated three times. The homogenate was centrifuged at 34500g for 10 min a t 4 "C. The pellet was repeatedly extracted by resuspension in cold PBS, homogenization, and centrifugation three more times. Each extraction step was sampled, and the remaining supernatant was combined with the supernatant of the previous extraction. The cell extract, medium extract, and shake flask broth were prepared for ion-exchange chromatography by filtration through a 0.22-pm syringe filter with a glass fiber prefilter. The total volume of the filtrate was recorded, as well as the pH and conductivity. The filtrates were diluted with 18 MQ water until the conductivity was less than 2 ma-'. Once diluted, the pH was checked and, if necessary, adjusted to 7.0 with either 2 N NaOH or HC1. The conductivity was then readjusted as needed until both specifications (conductivity and pH) were met. The extract was frozen a t -20 "C until needed. Thawing of the frozen material was performed in a lukewarm water bath while swirling gently. Partial purification of the filtrates was accomplished with a single ion-exchange step using the S-Sepharose fastflow resin from Pharmacia. A 6-mL column was used in these studies. Each chromatography run consisted of pouring a bed of virgin resin suspended in washing buffer (20 mM PBS, pH 7.0 and conductivity less than 2 m W ) and allowing the resin to settle. Once the flow adapter was in place, the bed was prepared according to the manufacturer's recommendations for cleaning. Next, the bed was washed for at least 30 min (5 column vol) with washing buffer. The column preparation was completed by checking the pH of the column effluent to be sure it was the same as that of the washing buffer. Once prepared, the column was loaded with the broth or cell extract. After the protein was loaded onto the column, the column was washed with washing buffer until the absorbance of the column effluent returned to the base-line absorbance seen prior to loading. The column was eluted with a linear gradient in ionic strength, typically from 0 to 200 mM NaCl in 20 mM PBS (pH 7.0) over 60 min using a flow rate of 1mL/min and lasting for 10column vol (1h for the 6-mL column). Two-milliliter fractions were collected during the column washing the elution. The effluent from the column was continually monitored with a Hitatchi Model 110 spectrophotometer set at 280 nm and equipped with a 20-pL flow-through cell. The absorbance was recorded by a chart recorder. The entire ion-exchange chromatography procedure was carried out at 4 "C in a cold room. Analytical Techniques. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to determine the molecular weights of the proteins in extraction and elution samples. Mini-Protean I1 precast gels (12 96 ) manufactured by Bio-Rad Laboratories were used. The samples were prepared by dilution with sample buffer containing mercaptoethanol and SDS. Purified trichosanthin, provided by Michael Piatak (Genelabs,Redwood City, CAI, was prepared in the same manner and used as a standard for comparison with the samples. The procedures used in sample loading, running, and silver staining the gels are described by Bio-Rad (1990a). The gels were
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Table 1. Results of A. rhizogenes Transformation Experiments period of period of results coculti- exposure to for vation antibiotic carrot controls (days) (weeks) results for 7'. kirilowii 4 7 plant tissue dead callus 4 8 all tissue healthy but no no callus callus observed 5 12 extensive callus from leaf, callus stem,and petiole 12 7 extensive callus from stem slight callus 14 12 callus from leaf callus
either silver-stained for detection of proteins or stained using an alcian blue glycoprotein stain followed by silver staining to detect proteins bound to acidic sugars. The Western blotting procedure was the most sensitive and definitive technique to positively identify trichosanthin. The primary antibody, provided by Genelabs, was a polyclonal antibody raised against GLQ223, the pharmaceutical preparation of trichosanthin. The secondary antibody, a rabbit anti-goat alkaline phosphatase conjugate, was obtained from Southern Biotechnology (Birmingham, AL). The blots were color-developed by incubation in the alkaline phosphatase substrates NBT and BCIP. Controls in which the primary antibody step was omitted were run for each blot to determine whether any of the bands were due to nonspecific binding of the secondary antibody. Confirmation of the presence of the rolb and rolc genes in the transformed cells was carried out using the polymerase chain reaction (PCR) method. Primers were designed from the available DNA sequence for A. rhizogenes A4 (Slightomet al., 1986). DNA was extracted from cells using the Dellaporta protocol (Dellaportaet al., 1983) and analyzed by PCR for rolb and rolc using a slight modification of the protocol that accompanies the AmpliTAq DNA polymerase from Perkin-Elmer Cetus (annealing and extension times were incresed to 2 min). Amplified DNA fragments were detected using DNA gel electrophoresis. High-voltagepaper electrophoresis was used to resolve opines in the tissue extract, and a silver-staining technique was used to stain the opines (Petit et al., 1983). Bioassays for RIP activity were performed using in vitro cell-free protein translation kits using treated rabbit reticulocyte (Promega, Madison, WI). The lysate (17.5 pL) was incubated with 10 pL of the sample for 15 min at room temperature. Following incubation with the samples, translation of luciferase mRNA was performed at 30 "C for 2 h using the exposed ribosomes. Finally, the luciferase activity was measured using a luminometer. RIP activity results are reported in terms of percent inhibition of protein synthesis relative to the control, which used 10 pL of 20 mM PBS buffer at pH 7.0.
Results and Discussion TransformationResults. Results of the A. rhizogenes transformation experimentsare presented in Table 1.None of the transformation attempts using either T. kirilowii or the carrot disks resulted in the hairy root phenotype, although several of the infections led to callus formation. For relatively short periods of cocultivation and antibiotic exposure, no tumorous growth of any kind was observed for T. kirilowii. However, for longer periods of cocultivation and/or exposure to the antibiotic, callus was obtained. The physical appearance of the T. kirilowii tissue controls that were not infected with the agrobac-
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Figure 1. PCR analysis of rolb and rolc genes. Lane descrip tions: (1) blank; (2) agrobacterium-transformed callus PCR for rolb; (3) agrobacterium-transformedcallus PCR for rolc; (4) 123bp ladder; (5) E. coli 113 PCR for rolb; (6) E. coli 113 PCR for rolc.
terium remained unchanged. Although the hairy root phenotype was never observed, it was evident from the stable growth (even after over 2 years of monthly subculturing) of the callus-like tissue on hormone-free medium that some portion of the transferable T-DNA from the agrobacterium had been incorporated into the plant genome. PCR analysis was used to detect the rolb and rolc genes in tenth generation agrobacteriumtransformed callus growing on hormone-free LinsmaierSkoog medium without antibiotics. The results are shown in Figure 1; lanes 2 and 3 show the expected 775- and 539-bp sequences for rolb and FOZC genes, respectively, for the plant callus, lane 4 contains a 123-bp ladder marker, and lanes 5 and 6 are the positive controls for rolb and rolc. There are also indications that the ability to form the transformed callus is tissue-dependent, since some of the infection protocols led to callus for some types of explant, while other tissue types were not affected. None of the transformation protocols were successful for the root tissue. An agropine assay was performed on the callus tissue generated from the carrot disks and T. kirilowii leaf, petiole, and stem. Agropine was not detected in any of the samples (data not shown). The mechanism of transformation for the A. rhizogenes A4 strain provides two possible explanations. Either the TR-DNAcontaining the ags genes responsible for the agropine synthesis was not transferred to the plant cells' genomes or, if the transfer did occur, the ags genes were not being expressed. Since it is frequently observed that the transformation process using A. rhizogenes A4 is variable (Le., only one of two T-DNA segments is incorporated into the plant's genome), the first explanation is most likely. Although the absence of TR-DNAwould account for the absence of the agropine, it is clear that at least the rolb and rolc genes have been transferred and that the plant cell phytohormone levels are being altered, causing the formation and stable growth of callus tissue on hormone-freemedium. These observations indicate that the TL-DNA,or a fragment of the TL-
Biotechnol. Rug., 1994, Vol. 10, No. 4
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Fraction Number Figure 2. Elution profile from ion-exchangepurification of the intracellular proteins from the agrobacterium-transformed T. kirilowii cells grown in shake flasks (-) and RIP activity of elution fractionsreported in terms of percent inhibition of protein synthesis using a rabbit reticulocyte system (m).
DNA, has been incorporated into the plant genome. We are currently performing additional experiments to determine the nature of the genetic transformation events. Although the transformation experiments did not produce the traditional "hairy root" phenotype, the agrobacteriumtransformed callus is very attractive from a bioprocessing standpoint, since it is much more easily cultured than hairy roots and eliminates the need for exogenously supplied hormones compared with nontransformed callus cultures. Agrobacterium-TransformedSuspension Cultures. Figures 2 and 3 show the elution profiles of the purified cell extract and broth, respectively. Figures 4 and 5 show silver-stained SDS-PAGE gels of representative elution fractions of the purified intracellular and extracellular samples. Figures 6 and 7 show Western blots (for trichosanthin) for the intracellular and extracellular proteins, respectively. During the gradient elution of the intracellular proteins, three somewhat distinct peaks were observed (Figure 2). The first peak (elution fractions 4-8) comprise two major proteins with molecularmasses of 21.5 and 33.5 kDa (lanes 2 and 3, Figure 4). As the second peak begins around elution fraction 10, we start to see a band just above the trichosanthin standard (lane 4, Figure 4) at about 29 kDa and a somewhat more diffuse band with the same relative mobility as the trichosanthin standard. The 29-kDa protein continues to elute during the second and third peaks (lanes 5,7, and 8, Figure 4), with the highest levels seen at peak 3 (lane 8, Figure 4). The band at the same relative mobility as trichosanthin appears to be the strongest in elution fractions 12-14, which correspond to the tail of peak 2 (lanes 5 and 7, Figure 4). The Western blot of elution fractions 12 and 14 confirms the presence of trichosanthin (lanes 2 and 3, Figure 6a). Comparison of the trichosanthin Western blot (Figure 6a) and the control blot (Figure 6b), in which the primary antibody step was omitted, indicates that the higher molecular weight immunoreactive bands at 38 kDa are the result of cross-reactivity to the trichosanthin antibody and are not due to nonspecificbinding. Other researchers have shown that antiserum to one RIP may be cross-reactive to RIPS fromthe same species (Irvin et al., 1980;Preston and Ervin, 1987). The gradient elution of the extracellular proteins shows a much larger amount of protein, with one major peak between elution fractions 6 and 18 and a much smaller peak at the end of the elution profile (Figure 3). The
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Figure 3. Elution profile from ion-exchangepurification of the shake flask broth from the agrobacterium-transformed 2'. kirilowii cultures (-) and RIP activity of elution fractions reported in terms of percent inhibition of protein synthesis using a rabbit reticulocyte system (v, 0).
+,
97.4kD 66.2kD 45kD 31kD
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Figure 4. Silver-stainedSDS-PAGEgel of intracellular proteins extracted and purified from agrobacterium-transformed 2'. kirilowii cells grown in suspension. Lane descriptions (amounts shown are actual volumes loaded and estimated protein mass based on an extinction coefficientof0.75AU/(mg/mL)): (1)blank; (2)fraction 6 (10pL, -750 ng); (3)fraction 8 (19pL, -1500 ng); (4)fraction 10 (19pL, -300 ng); (5)fraction 12 (19pL, -1300 ng); (6)trichosanthin standard (200ng); (7) fraction 14 (19pL, -600 ng); (8) fraction 16 (19pL, -600 ng); (9)Bio-Rad silverstained low molecular mass standards 97.4,66.2,45.0,31.0,and 21.5 kDa (10pL); (10)blank.
29-kDa protein elutes throughout most of the large peak; it is first seen in fraction 8 and extends through at least fraction 15(lanes 4-6, Figure 5). The 29-kDa protein isalso observed to be a major component in the smaller peak. Although a slight diffuse band at the same molecular weight as trichosanthin was observed on the SDS-PAGE gels, trichosanthin was not detected in the Western blot of these broth elution fractions (Figure 7). Since we loaded at least 1pg of protein onto lanes 3 and 4 and at least 200 ng of protein onto lanes 5 and 6, we would have detected trichosanthin if it made up at least 10%of the total protein in the sample. The fact that the 29" band is not present on the Western blot even though the loadingof this protein is at least 200 ng is not surprising since the trichosanthin antibody is not immunoreactive with the 29-kDa protein found in root tuber (TAP-29),and we have never observed any 29-kDa bands on Western blots using the trichosanthin antibody.
If we assume an average extinction coefficient of 0.75 AU/(mg/mL) a t 280 nm, the total protein content of the
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1 2 3 4 5 6 7 8 9 1 0 Figure 5. Silver-stained SDS-PAGE gel of extracellular proteins purified from suspension broth of agrobacterium-transformed T. kirilowii cultures. Lane descriptions (for elution fractions, the amounts shown are actual volumes loaded and estimated protein mass based on an extiction coefficient of 0.75 AU/(mg/ mL)): (1)wash fraction 1 (19pL); (2)wash fraction 9 (19pL); (3)fraction 4 (19pL, -2900 ng); (4)fraction 8 (4pL, -3500 ng); (5)fraction 11 (0.4pL, -600 ng); (6)fraction 15 (0.2pL, -400 ng); (7)trichosanthin standard (200ng); (8)fraction 19 (19pL, 2000 ng); (9)fraction 24 (8pL, 1500ng); (10)Bio-Rad silverstained low molecular mass standards 97.4,66.2,45.0,31.0,and 21.5 kDa (10pL).
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Figure 6. Trichosanthin Western blot (a, left) and control blot (b, right) of intracellular proteins extracted and purified from agrobacterium-transformedT. kirilowiicells grown in suspension. Lane descriptions (amounts shown are actual volumes loaded and estimated protein mass based on an extinction coefficient of 75 AU/(mg/mL): (1)fraction 10 (11pL, 150ng); (2)fraction 12 (11 pL, -700 ng); (3)fraction 14 (11 pL, -350 ng); (4) trichosanthinstandard (20ng); (5)Bio-Rad prestained molecular mass standards 106.0,80.0,49.5,32.5,27.5,and 18.5 kDa (7pL); (6-10)reverse order of lanes 1-5.
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first peak from the broth purification is estimated at 30 mg. If we assume that the 29-kDa protein accounts for at least one-half of the total protein in these elution fractions, the amount of the 29-kDa protein obtained from the broth of the agrobacterium-transformed cultures is approximately 1.1% of the dry weight of the callus (the dry weight of the callus was estimated as 1.4 g). This is most likely an underestimate of the 29-kDa protein levels in the broth for several reasons. First, this estimate assumes that the absorbance varies linearly with concentration over the entire 0-2 AU range, which results in an underestimation of the protein level. Second, given the large amount of
!7.5kD
1 2 3 4 5 6 7 8 9 10 Figure 7. Trichosanthin Western blot of extracellular proteins purified from the broth of agrobacterium-transformed2'. kirilowii cultures. Lane descriptions (amounts shown are actual volumes loaded and estimated protein mass based on an extinction coefficient of 0.75AU/(mg/mL)): (1)wash fraction 1 (19pL); (2) wash fraction 9 (10pL); (3)fraction 4 (10pL, -3000 ng); (4) fraction 8 (4pL, -1700 ng); (5)fraction 11 (0.4pL, -600 ng); (6) fraction 15 (0.2pL, -500 ng); (7)trichosanthin standard (20 ng); (8) fraction 19 (10pL, -2000 ng); (9)fraction 24 (8pL, -1600 ng); (10)Bio-Rad prestained molecular mass standards 106.0, 80.0,49.5,32.5, 27.5,and 18.5 kDa (7pL).
protein eluted, we may have exceeded the capacity of the column so that some of the 29-kDa protein passed through during loading. Root tuber obtained from a plant generated from the same seed source was also analyzed for comparison. An SDS-PAGE gel of proteins extracted from the root tuber showed approximately nine proteins ranging in molecular mass from 15.7 to 60.9 kDa (data not shown). A Western blot of elution fractions from the purified root tuber extract confirmed the presence of trichosanthin in the root tuber. The root tuber also contained a strong band that ran about 2 kDa above the trichosanthin band (presumably, this is TAP-29) that was not detected by the trichosanthin antibody, indicating that the trichosanthin antibody is not cross-reactiveto TAP-29. The alcian blue glycoprotein stain showed that trichosanthin was not glycosylated,while TAP-29 was glycosylated (data not shown). The yield of trichosanthin obtained from the root tuber is around 1.31.8% (w/w) of the dry weight of the root tuber, while the yield of TAP-29 is estimated to be 0.4-1.0% (w/w) of the dry weight of the root tuber. Although the highest yield of the 29-kDa protein was found extracellularly in the broth from the agrobacterium-transformed callus, a 29kDa band was also observed in purified extracts from nontransformed callus grown on semisolid medium, as well as cells grown in suspension. Figures 1and 2 also show the RIP activity results (in terms of percent inhibition of protein translation using a cell-free rabbit reticulocyte system) for the intracellular and extracellular elution fractions, respectively. Strong inhibition of protein translation is observed for the intracellular fractions 7-16, indicating the presence of one or more RIPS. Since trichosanthin was only detected in fractions 12 and 14, these results suggest the presence of other RIPSin these samples. SDS-PAGE gels of fractions in this range also contain the 29-kDa band (lanes 3-5 and 7-8, Figure 4); the 29-kDa protein may be responsible for the RIP activity. Analysis of RIP activity for the extracellular elution fractions shows that one or more of the proteins in the large peak comprising elution fractions 5-16 severely inhibit protein translation, most likely as the result of deactivation of the ribosomes. Although
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trichosanthin was not detected in Western blots of these samples, there appears to be an RIP present. Given the fact that other researchers have found new RIPSin plant cell cultures that were not found in the intact plant, it is certainly possible that the RIP activity is due to one or more proteins not previously identified. Since several of the elution fractions are fairly pure, containing predominantly the 29-kDa protein (lanes 5 and 6, Figure 5), and since TAP-29, a 29-kDa protein obtained from the root tuber, is known to be an RIP, it is very likely that the 29-kDa protein is an RIP. When elution fractions 8 and 15were diluted 100-foldprior to performing the RIP assays, protein translation did occur, with the diluted elution fraction 8 showing 25% inhibition of protein synthesis and the diluted elution fraction 15showing 58 % inhibition of protein synthesis. It is also interesting to note, however, that a strong 29-kDa band is observed in elution fraction 24 (lane 9, Figure 5), although essentially no RIP activity was observed for this fraction. It may be that these proteins, although very close in molecular weight, differ enough in composition to confer very different purification characteristics and biological activity. We are currently performing additional experiments to further characterize the proteins obtained from the intracellular and extracellular samples.
Buenviaje and Arlene Adela for assistance with the RIP bioassays, and Sandra Uratsu for performing the PCR work. K.M. gratefully acknowledges NSF support (Grant No. BCS-9010841) for this project.
Conclusions
Dellaporta, S. L.; et al. A plant DNA minipreparation: version 11. Plant Mol. Biol. Rep. 1983, 1, 19. Dixon, R. A. Plant cell culture, apractical approach; IRL Press: Washington, D.C., 1985. Endo, Y.; Tsurugi, K. RNA N-glycosidase activity of ricin A chain: Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J. Biol. Chem. 1987,262, 8128-8130. Endo, Y.; Tsurugi, K.The RNA N-Glycosidase activity of ricin A chain: characterization of the enzymatic activity of ricin A-chain with ribosomes and with rRNA. J.Biol. Chem. 1988,
Transformation using Agrobacterium rhizogenes A4 resulted in a callus that grows well on hormone-free medium. When grown in suspension culture, these cells intracellularly produce trichosanthin and excrete high levels of a 29-kDa protein, which is thought to be TAP-29 or an isoform of this protein. Current studies are underway to further characterize this protein, the higher molecular weight proteins found in the broth, and the nature of the transfer of genetic material. Although the exact nature of the genetic transfer is not yet known, transfer of the rolb and rolc genes was confirmed. The fact that the transformed cells have been maintained on hormone-free medium for over 2 years indicates that the transformation has altered levels of endogenous hormones. Since it is known that hormone levels have a strong influence on RIP production in culture (Barbieri, 1989), the transformation may have altered the regulation of RIP gene expression because trichosanthin was found intracellularly in the transformed cells but was not detected in the nontransformed cells. Such plant cell systems may be useful as a mechanism of de nouo synthesis of proteins or RIP isoforms that are not expressed in the intact plant. From a bioprocessing standpoint, the tumorous cell line offers a number of potential advantages over alternative methods of RIP production. Efficient excretion of RIPS into the broth simplifies purification and allows for the possibility of in situ product removal. Furthermore, our preliminary studies indicate that purification of the 29kDa protein from the broth would be relatively straightforward. The tumorous callus grows well on hormonefree medium, thereby eliminating the cost of synthetic hormones and the tedious experimentation required to optimize hormone types and levels if exogenouslysupplied. Compared with hairy root cultures, callus is much easier to work with in a bioreactor, allowing for easier inoculation, sampling, and mixing. Further work is necessary to assess the relative advantages with respect to growth rates, genetic stability, and stability of production rates over time.
Acknowledgment The authors thank Mike Piatak at Genelabs (Redwood City, CA) for providing technical assistance, Cynthia
Literature Cited Barbieri, L.; Bolognesi, A.; Cenini, P.; Falasca, A.I.; Minghetti, A.; Garofano,L.; Guicciardi, A.;Lappi, D.; Miller, S. P.; Stirpe, F. Ribosome-inactivating proteins from plant cells in culture. Biochem. J. 1989,257,801-807. Bio-Rad LaboratoriesInstruction manual: Mini-PROTEAN ZZ Dual Slab Cell; Bio-Rad Labs: Richmond, CA, 1990. Bonneas, M. S.; Mabry, T. J. Tissuecultureof endod (Phytolacca dodecandra L-Herit): Growth and production of ribosome inactivating proteins. Plant Cell Rep. 1992,11, 66-70. Byers, V. S.; Levin, A. S.; Waites, L. A.; Starrett, B. A.;Mayer, R. A.; Clegg, J. A.; Price, M. R.; Robins, R. A.; Delaney, M.; Baldwin, R. W. A phase 1/11study of trichosanthin treatment of HIV disease. AIDS 1990,4, 1189-1196. Chikon,M.-D.; Tepfer, D. A.; Petit, A.; David, C.; Casse-Delbart, F.; TempB, J. Agrobacterium rhizogenes inserts T-DNA into the genome of the host plant root cells. Nature 1982,295, 432-434.
Dandekar, A. M.; McGranahan, G. H.; Leslie, C. A.; Uratsu, S. L. Agrobacterium-mediated transformation of somatic embryos as a method for the production of transgenic plants. J. Tissue Culture Methods 1989, 12, 145-150.
263, (18), 8735-8739.
Endo, Y.; Mitsui, K.; Motizuki, M.; Tsurugi, K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. J. Biol. Chem. 1987, 262, 5908-5912. Endo, Y.; Tsurugi, K.; Yutsudo, T.; Takeda, Y.; Ogasawara, T.; Igarashi, K. Site of action of a Vero toxin (VT2) from Escherichia coli 0157:H7 and of a Shiga toxin on eukaryotic ribosomes. Eur. J. Biochem. 1988,171, 45-50. Estruch, J. J.; Chriqui, D.; Grossman, K.; Schell, J.; Spena, A. The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates. EMBO J. 1991a, 10, 2889-2895.
Estruch, J. J.; Schell, J.; Spena, A. The protein encoded by the rolB plant oncogene hydrolyses indole glucosides. EMBO J. 1991b, 10, 3125-3128.
Flores, H. E.; Hoy, M. W.; Pickard, J. J. Secondary metabolites from root cultures. TZBTECH 1987,5, 64-69. Gheysen, G.; Dhaese, P.; Van Montagu, M.; Schell, J. DNA flux across genetic barriers: The crown gall phenomenon. In Genetic Flux in Plants;Hohn, B., Dennis, E. S.,Eds.;SpringerVerlag: Vienna, 1985; pp 11-47. Harley, M. R.; Legname, G.; Osborn, R.; Chen, Z.; Lord, J. M. Single-chainribosome inactivating proteins from plants depurinate Escherichia coli 235 ribosomal RNA. FEBS 1991,290, 65-68.
Ikeda, T.; Takanami, Y.; Imaizumi, S.; Matsumoto, T.; Mikami, Y.; Kubo, S. Formation of anti-plantviral protein by Mirabilis jalapa L. cells in suspension culture. Plant Cell Rep. 1987a, 6,216-218.
Ikeda, T.; Niino, K.; Kataoka, J.; Matsumoto, T. Effects of nutritional and hormonal factors on the formation of antiplant-viralprotein by Mirabilis jalapa L. cells in suspension culture. Agric. Biol. Chem. 1987b,51, 3119-3124. Ikeda, T.; Niino, K.; Kataoka, J.; Matsumoto, T. Effects of phosphate concentration on growth in Mirabilis jalapa cultured cells. Agric. Biol. Chem. 1987c, 51, 2611-2612. Ikeda, T.; Niino, K.; Kataoka, J.; Matsumoto, T. Effects of culture conditionson the formation of anti-plant-viralproteinby highly
Biotechnol. Prog., 1994, Vol. 10, No. 4
352
producingcellstrains of Mirabilis jalapa L. Agric. Biol. Chem. 1988a, 52,2611-2613.
Ikeda, T.; Kataoka, J.; Konno, Y.; Imaizumi, S.; Kuwata, S.; Takanami, Y.; Mataumoto,T. Selectionof Cultured Mirabilis jalapa L. cells: strains producing high levels of anti-plant viral proteins. Agric. Biol. Chem. 1988b, 52, 1383-1388. Irvin, J. D.; Kelly, T.; Robertus, J. D. Arch. Biochem. Biophys. 1980,200,418-425.
Kahn,J. 0.;Kaplan, L. D.; Gambertoglio, J. G.; Bredesen, D.; Arri, C. J.; Turin, L.; Kibort, T.; Williams, R.L.; Lifson,J. D.;
Volberding, P. A. Thesafetyand pharmacokinetics of GLQ223 in subjects with AIDS and AIDS-related complex: a phase I study. AIDS 1990,4,1197-1204. Lee-Hung, S.;Huang, P. L.; Kung, H.-F.; Li, B.-Q.; Huang, P. L.; Huang, P.; Huang, H. I.; Chen, H.4. TAP29 An antihuman immunodeficiencyvirus protein from Trichosanthes kirilowiithatisnontoxictointactcells. R o c . Natl. Acad. Sci. U.S.A. 1991,88,6570-6574.
McGrath, M. S.; Hwang, K. M.; Caldwell, S. E.; Gaston, I.; Luk, K. C.; Ledas, P. V.; Vennari, J. C.; Yeung, H. W.; Lifson,J. D. An inhibitor of human immunodeficiencyvirus replication in acutely and chronically infected cells of lymphocyte and mononuclearphagocytelineage. h o c . Natl. Acad. Sci. U.S.A. 1989,86,2844-2848. Moore, L.; Warren, G.; Strobel, G. Involvement of a plasmid in the hairy root disease of planta caused by Agrobacterium rhizogenes. Plasmid 1979,2,617-626. Ng,T.B.;Chan, W. Y.;Yeung,H. W.Proteinswithabortifacient,
ribosome inactivating, immunomodulatory, antitumor and anti-AIDS activities from Cucurbitaceae plants. Gen. Pharmacol. 1992,23,575-590.
Petit, A,; David, C.; Dahl, G. A,; Ellis, J. G.; Guyon, P.; CasseDelbart, F.; Tempe, J. Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol. Gen. Genet. 1983,190,204-214. Preston, J. F., III; Ervin, S. E. Fed. Proc. 1987,46, 2083. Savary, B. J.; Flores, H. Production of extracellular proteins by root cultures of Chinese medicinal cucumber (Trichosanthes spp), Abstract, American Society of Plant Physiologists, Albuquerque, NM, July 28-Auguat 1,1991. Slightom, J. L.; Durand-Tradif, M.; Jouanin, L.; Tepfer, D. J. Nucleotide sequence analysis of TL-DNA of Agrobacterium
rhizogenes agropine type plasmid Identification of open reading frames. J. Biol. Chem. 1986,261, 108. Spano, L.; Pomponi, M.; Constantino, P.; Van Slogteren, G. M. S.; Temp& J. Identification of T-DNA in the root-inducing plasmid of the agropine type Agrobacterium rhizogenes 1855. Plant Mol. Biol. 1982,1, 291-300.
Stirpe,F.;Barbieri, L. Ribosome-inactivatingproteins up to date. FEBS Lett. 1986,195, 1-8.
Stirpe, F.; Barbieri, L.; Battelli, M. G.; Soria, M.; Lappi, D. A. Ribosome-inactivating proteins from plants: Present status and future prospects. Bioltechnology 1992,10, 405-412. Tepfer, D. Genetic transformation using Agrobacterium rhizogenes. Physiol. Plant. 1989, 79, 140-146.
Thomsen, S.; Hansen, H. S.; Nyman, U. Ribosome-inhibiting proteins from in vitro cultures of Phytolacca dodecandra. Planta Med. 1991,57,232-236.
Uratsu, S . L.; Amadhi, H.; Bringhurst, R. S.; Dandekar, A. M. Relative virulence of Agrobacterium strains on Strawberry (Fragaria vesca). Hort. Science 26, 196-199.
White, F. F.; Nester, E. W. Hairy root: Plasmid encodes virulence traits in A. rhizogenes. J. Bacteriol. 1980a, 141,1134-1141. White, F. F.; Nester, E. W. Relationship of plasmids responsible for hairy root and crown gall tumorigenicity. J. Bacteriol. 1980b, 141, 710-720.
White, F. F.; Ghidossi, G.; Gordon, M. P.; Nester, E. W. Tumor induction by Agrobacterium rhizogenes involves the transfer of plasmid DNA to the plant genome. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 3193-3197.
White, R. R.; Taylor, B. H.; Huffman, G. A.; Gordon, M. P.; Nester, E. W. Molecular and geneticanalysisof the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J. Bacteriol. 1985, 164, 33-44.
Willmitzer,L.; Sanchez-Serrano,J.;Buschfeld,E.; Schell,J. DNA fromAgrobacterium rhizogenes istransferred to and expressed in hairy root plant tissues. Mol. Gen. Genet. 1982,186,16-22. Accepted November 23, 1993.' ~~
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Abstract published in Advance ACS Abstracts, March 15,1994.
