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Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures
Protein Expression and PuriWcation 47 (2006) 68–73 www.elsevier.com/locate/yprep
Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures Shin-Young Hong a, Tae-Ho Kwon a, Yong-Suk Jang b, Soo-Ho Kim c, Moon-Sik Yang a,¤ a
Division of Biological Sciences and Research Institute for Bioactive Materials, Chonbuk National University, Jeonju 561-756, Republic of Korea b Bank for Cytokine Research, Chonbuk National University, Jeonju 561-756, Republic of Korea c Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju 561-756, Republic of Korea Received 8 August 2005, and in revised form 29 September 2005 Available online 2 November 2005
Abstract Human granulocyte-colony stimulating factor (hG-CSF), a human cytokine, was expressed in transgenic rice cell suspension culture. The hG-CSF gene was cloned into the rice expression vector containing the promoter, signal peptide, and terminator derived from a rice -amylase gene Amy3D. Using particle bombardment-mediated transformation, hG-CSF gene was introduced into the calli of rice (Oryza sativa) cultivar Dong-jin. Expression of the hG-CSF gene was conWrmed by ELISA and Northern blot analysis. The amount of recombinant hG-CSF accumulated in culture medium from transgenic rice cell suspension culture on the sugar starvation was determined by time series ELISA. Biological activity of the plant derived hG-CSF was conWrmed by measuring the proliferation of the AML-193 cells, and was similar to that of the commercial Escherichia coli-derived hG-CSF. In this paper, we discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant hG-CSF. © 2005 Elsevier Inc. All rights reserved. Keywords: hG-CSF; Ramy3D; Rice cell suspension culture
Many proteins of therapeutic or scientiWc interest are expressed as recombinant proteins in heterologous cell cultures. Although recombinant proteins have typically been produced in microbial or animal cell cultures, plant cell cultures are also used for this purpose. Plant cells are now considered as viable and competitive expression systems for production of bioactive recombinant proteins of commercial value. Although product contamination by mycotoxins and plant secondary metabolites is a potential issue, these systems have several advantages over either prokaryotic or animal cell expression system [1]. For example, plant cellderived transgenic proteins are likely to be safer for human use than those derived from mammalian cells, since plant cell contaminants and viruses are not pathogenic to humans. A plant cell system is able to carry out the post translational modiWcations whereas prokaryotic system
*
Corresponding author. Fax: +82 63 270 4334. E-mail address: [email protected] (M.-S. Yang).
1046-5928/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.09.028
cannot. In addition, sexual crossing can generate multiple transgenic plants. Additionally, plant cells are generally inexpensive to grow on a large scale, and their production is not limited to fermentor capabilities. Furthermore, it is much easier and more economical to purify secreted foreign proteins from plant cell media than from complex mammalian cell media. Therefore, plant cell culture systems may be the most suitable means of producing small-to-medium quantities of high-priced, high purity, specialty recombinant proteins. Human granulocyte-colony stimulating factor (hGCSF)1 was a member of the long chain family of cytokines, Wrst reported by Nagata et al. [2]. The mature hG-CSF is a 19.6 kDa glycoprotein with 174 amino acids and is produced mainly by monocytes and macrophages upon activation by endotoxin [3]. G-CSF plays a critical role in the
1 Abbreviations used: hG-CSF, human granulocyte-colony stimulating factor; HPT, hygromycin phosphotransferase.
S.-Y. Hong et al. / Protein Expression and PuriWcation 47 (2006) 68–73
process of hematopoiesis, regulating the proliferation, diVerentiation, and survival of neutrophils and neutrophilic progenitor cells [4]. G-CSF has been expressed in various foreign hosts such as Escherichia coli [5], yeast [6], and mammalian cells [7] and is now employed to treat cancer patients undergoing chemotherapy to alleviate the depression of white blood cell levels produced by cytotoxic therapeutic agents. We previously produced hG-CSF using transformed tobacco cells in suspension cultures, and demonstrated that it was secreted into the culture medium in a bioactive form [8]. The amount of hG-CSF present in the culture medium increased over time to a maximum of 105 g/L at day 9 after incubation and dropped quickly. To overcome this problem, we used the rice amylase expression system Ramy3D [9] to manufacture recombinant hG-CSF in rice cell suspension culture, and found that the yield of hG-CSF was signiWcantly higher than that obtained in our previous transgenic tobacco expression system. Materials and methods Expression vector construction The hG-CSF cDNA was synthesized using RT-PCR technique with poly(A)+ mRNA from TPA stimulated THP-1 cell line. A pair of primers (5⬘-AAA GTA CTC CTC TGG GTC CTG CTA GCT C-3⬘ and 5⬘-TTG GTA CCC TTG GCT CAG GGC TGG-3⬘) were designed to generate the 555 bp of PCR fragment containing an open reading frame for hG-CSF lacking its signal peptide [2]. The PCR product was cloned into pUC18 vector (TAKARA, Japan) to generate the pMYO83. The DNA fragment containing the Ramy3D promoter and signal
69
peptide coding sequence [9] was ampliWed with primers 5⬘GAG CAT GCA CCA CCT GTG CTA GCT ACT CCA CTG-3⬘ and 5⬘-AAC TGC AGG CTT GAC CCG AGT TAC AGG TC-3⬘. The PCR product was digested with SphI and PstI, and subcloned into the pUC18 multiple cloning site. The resultant plasmid, pMYO83, was digested with ScaI and KpnI to excise the gene for hG-CSF, which was subcloned into the PstI (blunted with T4 DNA polymerase) and KpnI site of pMYN27. The resulting plasmid, pMYO85, was digested with HindIII and EcoRI, and a DNA fragment containing hG-CSF with the Ramy3D promoter and signal peptide coding sequence was inserted into the binary vector (Fig. 1). Transformation and screening of transgenic rice calli Rice calli were prepared and transformed using the particle bombardment mediated transformation previously described [10]. After bombardment with expression vectors of hG-CSF gene, callus were transferred to selection medium N6 [11] supplemented with 2,4-dichlorophenoxyacetic acid (2 mg/L), sucrose (30 g/L), proline (0.5 g/ L), glutamine (0.5 g/L), casein enzymatic hydrolysate (0.3 g/L), and hygromycin B (35 mg/L) every 2–3 weeks. Calli resistant to hygromycin B were grown in N6 media minus sucrose (N6 ¡ S) for 7 days and then analyzed for hG-CSF in transgenic calli by ELISA. Calli were then ranked according to the hG-CSF expression level. Culturing on the same media for 2 months, with subculturing every 15 days, further established the suspension culture with selected calli. A single small healthy, oV-white, compact, and fast growing callus with doubling time about 7 days from each transformed line was grown on N6 medium for 10–15 days.
Fig. 1. Schematic diagram of the gene construct utilized in this study. Transferred-DNA (T-DNA) of the Wnal plasmid is shown. RB, T-DNA right border; 3⬘ UTR, 3⬘ untranslated region of rice -amlyase gene; du35S, CaMV35S promoter with a duplicated enhancer region; HPT, hygromycin phosphotransferase; Tnos, terminator of nopaline synthase; and LB, T-DNA left border.
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Northern and Western blot analyses For Northern blot analysis, total RNA was isolated from suspension cells grown in N6 + S and N6 ¡ S liquid media by using the RNeasy plant total RNA mini kit (Qiagen, CA, USA). Northern blot analysis was conducted according to the procedure of Sambrook et al. [12]. The blots for Northern blot analysis were hybridized with [32 P]-labeled random-primed DNA using about 555 bp ScaI and PstI fragment from pMYO83 that included hG-CSF gene. For Western blot analysis, samples were electroblotted onto Hybond C Extra membrane (Amersham–Pharmacia Biotechnology, UK) after SDS–PAGE. Rat monoclonal anti human G-CSF antibody (Pharmingen, CA) at 1:2000 and HRP-conjugated goat anti-rat IgG (Sigma, MO) at 1:7000 were used as primary and secondary antibodies, respectively. Measurement of quantitative analyses and biological activity of hG-CSF After production of recombinant hG-CSF was induced incubating transgenic rice cells in N6 ¡ S liquid medium, the suspension culture was centrifuged at 12,000g for 10 min. One millilitre sample of the resulting culture supernatant was dialyzed against PBS overnight at 4 °C, and used to measure the quantitative analyses and biological activity of hG-CSF. The concentration of rhG-CSF in the cultured media was determined by hG-CSF speciWc ELISA kit (Endogene, Woburn, MA) according to the procedure provided by manufacturer. To determine the biological activity of hG-CSF produced by rice cell suspension culture, minimal level of the sample for the induced proliferation of hG-CSF required AML-193 cells was measured [13]. BrieXy, growth-factor-starved 1 £ 105 cells were suspended in 100 L of RPMI 1640 medium (Sigma, MO) supplemented with 5% FBS (HyClone Laboratories, Logan, UT, USA). The 25 ng of hG-CSF produced from O85-4 cell line and E. coli derived hG-CSF were used for biological activity analysis. After 27 h incubation, 1 Ci of [methyl-3H]thymidine (Amersham Lifescience, NJ, USA) was added into each well and incubated for an additional 32 h. The cells were harvested with cell harvester (Inotech, Switzerland) and the tritium content was measured with a liquid scintillation counter (Packard, USA). In these experiments, a recombinant E. coli-derived human G-CSF, which was purchased from Pharmingen (San Diego, CA, USA), was used as a standard.
DNA sequence analysis using the dideoxynucleotide chain termination method. The structure of the hG-CSF expression vector pMYO85, which contains the hygromycin phosphotransferase (HPT) gene as a selection marker. The hG-CSF gene was placed under the control of the Ramy3D promoter, expression can be induced by removing the sucrose from the culture (Fig. 1). Rice calli were transformed with the pMYO85 plasmid by particle bombardment. Four weeks after bombardment transformation, hygromycin resistant rice callus colonies were generated. Preliminary screening of 27 putative transgenic cells for detection of hG-CSF expression was conducted by Western blot analysis. To conWrm the integration of hG-CSF in the transgenic rice cell lines, genomic DNA PCR analysis was performed with primers designed to amplify the hG-CSF gene (data not shown). Expression of recombinant hG-CSF gene and protein in transgenic rice suspension cells ELISA was conducted to initially screen hG-CSF protein expression in the conditioned media and cell extracts of transgenic cell suspension. As shown in Fig. 2, hG-CSF levels in the transgenic callus lines, which were measured at day 7 after transfer to sucrose-free plate, varied widely. Line O85-4 produced the highest level of recombinant hGCSF (185 g/g cells). This resulted in identiWcation of four ‘high-producer’ lines (O85-2, -4, -5, -7), which were selected for further analysis. Transcripts of recombinant hG-CSF gene expression in these lines were detected by Northern blot analysis. Similarly hG-CSF transcript signals were detected in transgenic cell lines under sucrose-starvation conditions (Fig. 3). The hG-CSF mRNA was not detected in wild-type cell line. As shown in Fig. 4A, levels of hG-CSF mRNA in O85-4 increased until day 7 after induction and then decreased slowly. A little hG-CSF mRNA was expressed by day 7 in sucrose medium. There was little sucrose in culture medium after day 7 in growth phase due to consumption by rice cells,
Results IdentiWcation and characterization of transgenic rice suspension cell lines The cDNA clone of hG-CSF was synthesized using the RT-PCR technique from TPA stimulated THP-1 cell line. The nucleotide sequence of hG-CSF was conWrmed by
Fig. 2. Screening of transformed callus lines expressing high levels of hGCSF. The amount of hG-CSF was determined by ELISA as described in Materials and methods. Lane NC is negative control which used suspension cells transformed with vector only. Lanes 1–8 represent the transgenic rice cell lines expressing the hG-CSF selected from the previous genomic DNA PCR analysis (data not shown).
S.-Y. Hong et al. / Protein Expression and PuriWcation 47 (2006) 68–73
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band of »20 kDa. The molecular weight of the hG-CSF was similar to that of the E. coli-derived hG-CSF. The hGCSF was not detectable in wild-type cell lines according to SDS–PAGE or Western blot analysis. QuantiWcation and functional assay for recombinant hG-CSF Fig. 3. Northern blot analysis to determine expression of hG-CSF gene during rice suspension cultures without sucrose. Each lane was loaded with 10 g total RNA from non-transgenic and transgenic rice cells. Loading standards were indicated by ethidium bromide stained-rRNA in lower panel. Lane NC is negative control which used suspension cells transformed with vector only. Lanes 1–4 represent the transgenic rice cell lines expressing the hG-CSF selected from the previous ELISA data.
Fig. 4. (A) Northern blot analysis to determine the temporal expression pattern of the hG-CSF gene during plant cell suspension culture. NC is negative control which used suspension cells transformed with vector only. S+ and S¡ indicate media with and without sucrose, respectively. Loading standards are indicated by ethidium bromide-stained rRNA in the lower panel. (B) SDS–PAGE, and (C) Western blot analysis of hGCSF in culture medium. Lanes M, PC, and NC denote prestained molecular weight standards, positive control hG-CSF derived from E. coli, and negative control culture medium from suspension cells transformed with the parent vector only, respectively. Lane 1 represents the total secreted protein obtained from the culture supernatant at day 7 in the N6 + S media. Lanes 2–5 indicate the total secreted protein obtained from the culture supernatant at days 1, 5, 11, and 15 in the N6 ¡ S media, respectively.
which induced the Ramy3D expression system. Levels of recombinant hG-CSF protein accumulated in culture medium were determined by Western blot analysis (Fig. 4C). In Fig. 4C, there were faint bands in lanes 1 and 2, which corresponded to day 7 in the growth phase and day 1 in the induction phase, respectively. The non-glycosylated E. coliderived recombinant hG-CSF appeared as a band at »20 kDa on the Western blot analysis. The culture medium from the transgenic rice cells produced hG-CSF protein
For the amount of hG-CSF in culture medium, results of time course experiments following total secreted protein and recombinant hG-CSF production are depicted in Fig. 5. ELISA was conducted for quantifying the secreted hG-CSF proteins in the suspension cells. The level of recombinant protein increased sharply in the production phase to a maximum of 2.5 mg/L at day 13. Recombinant hG-CSF comprised about 0.7% of the total secreted protein (Fig. 5). The biological activity of recombinant hG-CSF produced in transgenic rice cells was measured with E. coliderived hG-CSF, on AML-193 cells (Fig. 6). The culture
Fig. 5. Time course of hG-CSF and total secreted protein accumulated in culture medium of suspension cultures as determined by ELISA and Bradford assay, respectively. Rice cells in suspension culture were propagated for 9 days in N6 + S media, transferred to N6 ¡ S media, and cultured for the lengths of time (days) indicated.
Fig. 6. SpeciWc activity of recombinant hG-CSF produced from transgenic rice suspension cells. The 25 ng/ml of plant derived hG-CSF (phG-CSF) and E. coli-derived hG-CSF (PC) were used for biological activity analysis. NC is negative control which used culture medium of suspension cells transformed with vector only.
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system is a suitable system for production of valuable recombinant proteins combined with the advantages of plant cell culture.
medium from rice cells transformed with the parent vector only was used as a negative control, it did not support the growth of hG-CSF required AML-193 cells. The recombinant hG-CSF produced from the O85-4 cell line was eVectively dependent on the proliferation of AML-193 cells. The biological activity of the recombinant hG-CSF tested here matched completely with the data of ELISA, which demonstrated that it was correctly folded and functional. The speciWc activity of recombinant hG-CSF produced from transgenic rice cell suspension culture was similar to that exhibited by the commercial E. coli-derived hG-CSF.
This research was supported by a grant (Next Generation New Technology Development Project) from MOCIE, Republic of Korea. Dr. Soo-Ho Kim was supported by the post-doctoral program of Chonbuk National University, Republic of Korea.
Discussion
References
In this study, we have characterized the suitability of genetically engineered rice cell suspension culture system for the expression of recombinant human G-CSF. Human G-CSF have previously been expressed and secreted into culture media in a biologically active form [8]. The amount of hG-CSF produced and secreted by cultured transgenic tobacco suspension was about 105 g/L in 9 days after the initiation of the cell suspension culture. After 9 days, however, the rapid decrease in the amount of recombinant protein in the culture medium was observed. Consistent with our Wndings, previous reports suggests that, when present in plant cell culture medium, the stability and accumulation of recombinant proteins may decrease [14,15]. For this study, improving accumulation of target proteins in cell culture media by the use of protein stability agents such as PVP, gelatin and mannitol [16] has been explored. Hence, we opted to use a sucrose-starvation inducible, -amylase 3D promoter system. When rice amylase genes are expressed in the germinating seed, the Ramy3D was expressed in rice culture cells during sugar starvation. Terashima et al. [17] and Stoger et al. [18] demonstrated observable eVects in expression of pharmaceutical proteins in plant bioreactor systems, using Ramy3D promoter. We have shown previously that under the control of Ramy3D signal peptide, recombinant human granulocyte–macrophage colony stimulating factor were secreted into the cell culture medium [19]. We also found that protease activity in the culture medium upon induction of the Ramy3D promoter by sucrose starvation was very low. By exploiting this expression system, we increased the production of hG-CSF compared with our previous expression system, which utilized tobacco cell suspension cultures and the CaMV 35S promoter. The amount of recombinant protein in the medium increased until 13 days after induction, after which it dropped quickly. The maximum amount of hG-CSF was obtained in the rice cell culture medium with the Ramy3D expression systems. We suggest, based on this study that the current transgenic rice cell suspension culture system, especially Ramy3D, can be further developed as an experimental bio-reactor system for evaluating and upgrading the production of hG-CSF proteins in plant system. We therefore believe that the Ramy 3D expression
[1] S. Hellwig, J. Drossard, R.M. Twyman, R. Fischer, Plant cell cultures for the production of recombinant proteins, Nat. Biotechnol. 22 (2004) 1415–1422. [2] S. Nagata, M. Tsuchiya, S. Asano, Y. Kaziro, T. Yamazaki, O. Yamamoto, T. Hirata, N. Kubota, M. Oheda, H. Nomura, M. Ono, Molecular cloning and expression of cDNA for human granulocytecolony stimulating factor, Nature 319 (1986) 415–418. [3] J.E. Rasko, N.M. Gough, Granulocyte–macrophage colony-stimulating factor, in: A. Thomson (Ed.), The Cytokine Handbook, Academic Press, London, 1994. [4] L.M. Souza, T.C. Boone, J. Gabrilove, P.H. Lai, K.M. Zsebo, D.C. Murdock, V.R. Chazin, J. Bruszewski, H. Lu, K.K. Chen, Recombinant human granulocyte-colony stimulating factor: eVects on normal and leukemic myeloid cells, Science 232 (1986) 61–65. [5] J. Perez-Perez, C. Martinez-Caja, J.L. Barbero, J. Gutierrez, DnaK/ DnaJ supplementation improves the periplasmic production of human granulocyte-colony stimulating factor in Escherichia coli, Biochem. Biophys. Res. Commun. 210 (1995) 524–529. [6] C.S. Bae, D.S. Yang, K.R. Jang, B.L. Seong, J. Lee, Enhanced secretion of human granulocyte-colony stimulating factor directed by a novel hybrid fusion peptide from recombinant Saccharomyces cerevisiae at high cell concentration, Biotechnol. Bioeng. 57 (1998) 600–609. [7] M. Tsuchiya, H. Nomura, S. Asano, Y. Kaziro, S. Nagata, Characterization of recombinant human granulocyte-colony stimulating factor produced in mouse cells, EMBO J. 6 (1987) 611–616. [8] S.Y. Hong, T.H. Kwon, J.H. Lee, Y.S. Jang, M.S. Yang, Production of biologically active hG-CSF by transgenic plant cell suspension culture, Enzyme Microb. Technol. 30 (2002) 763–767. [9] K. Toyofuku, T. Umemura, J. Yamaguch, Promoter elements required for sugar-repression of the RAmy3D gene for alpha-amylase in rice, FEBS Lett. 428 (1998) 275–280. [10] L. Chen, P. Marmey, N.J. Taylor, J.P. Brizard, C. Espinoza, P. D’Cruz, H. Huet, S. Zhang, A. de Kochko, R.N. Beachy, C.M. Fauquet, Expression and inheritance of multiple transgenes in rice plants, Nat. Biotechnol. 11 (1998) 1060–1064. [11] C.C. Chu, C.C. Wang, C.S. Sun, C. Hsu, K.C. Yin, C.Y. Chu, F.Y. Bi, Establishment of an eYcient medium for anther culture of rice through comparative experiments on the nitrogen sources, Sci. Sin. 18 (1975) 659–668. [12] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory Press, NewYork, 1989. [13] A.L. Abegg, L.E. Vickery, M.E. Bremer, A.M. Donnelly, P.D. Doshi, M.L. Evans, T.L. Thurman, S.R. Braford, M.H. Caparon, S.C. Bauer, J.G. Giri, J.K. Welply, J.P. McKearn, W.G. Smith, The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist, Leukemia 16 (2002) 316–326. [14] J.M. Sharp, P.M. Doran, Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures, Biotechnol. Prog. 17 (2001) 979–992. [15] B.M. Tsoi, P.M. Doran, EVect of medium properties and additives on antibody stability and accumulation in suspended plant cell cultures, Biotechnol. Appl. Biochem. 35 (2002) 171–180.
Acknowledgment
S.-Y. Hong et al. / Protein Expression and PuriWcation 47 (2006) 68–73 [16] N.S. Magnuson, P.M. Linzmaier, J.W. Gao, R. Reeves, G. An, J.M. Lee, Enhanced recovery of a secreted mammalian protein from suspension culture of genetically modiWed tobacco cells, Protein Expr. Purif. 7 (1996) 220–228. [17] M. Terashima, Y. Muri, M. Kawamura, S. Nkanishi, T. Stolz, L. Chen, W. Drohan, R.L. Rodriguez, S. Katoh, Production of functional human 1-antitrypsin by plant cell culture, Appl. Microbiol. Biotechnol. 52 (1999) 516–523.
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[18] E. Stoger, C. Vaquero, E. Torres, M. Sack, L. Nicholson, J. Drossard, S. Williams, D. Keen, Y. Perrin, P. Christou, R. Fischer, Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies, Plant Mol. Biol. 42 (2000) 583–590. [19] Y.J. Shin, S.Y. Hong, T.H. Kwon, Y.S. Jang, M.S. Yang, High level of expression of recombinant human granulocyte–macrophage colony stimulating factor in transgenic rice cell suspension culture, Biotechnol. Bioeng. 82 (2003) 778–783.
Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures Shin-Young Hong a, Tae-Ho Kwon a, Yong-Suk Jang b, Soo-Ho Kim c, Moon-Sik Yang a,¤ a
Division of Biological Sciences and Research Institute for Bioactive Materials, Chonbuk National University, Jeonju 561-756, Republic of Korea b Bank for Cytokine Research, Chonbuk National University, Jeonju 561-756, Republic of Korea c Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju 561-756, Republic of Korea Received 8 August 2005, and in revised form 29 September 2005 Available online 2 November 2005
Abstract Human granulocyte-colony stimulating factor (hG-CSF), a human cytokine, was expressed in transgenic rice cell suspension culture. The hG-CSF gene was cloned into the rice expression vector containing the promoter, signal peptide, and terminator derived from a rice -amylase gene Amy3D. Using particle bombardment-mediated transformation, hG-CSF gene was introduced into the calli of rice (Oryza sativa) cultivar Dong-jin. Expression of the hG-CSF gene was conWrmed by ELISA and Northern blot analysis. The amount of recombinant hG-CSF accumulated in culture medium from transgenic rice cell suspension culture on the sugar starvation was determined by time series ELISA. Biological activity of the plant derived hG-CSF was conWrmed by measuring the proliferation of the AML-193 cells, and was similar to that of the commercial Escherichia coli-derived hG-CSF. In this paper, we discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant hG-CSF. © 2005 Elsevier Inc. All rights reserved. Keywords: hG-CSF; Ramy3D; Rice cell suspension culture
Many proteins of therapeutic or scientiWc interest are expressed as recombinant proteins in heterologous cell cultures. Although recombinant proteins have typically been produced in microbial or animal cell cultures, plant cell cultures are also used for this purpose. Plant cells are now considered as viable and competitive expression systems for production of bioactive recombinant proteins of commercial value. Although product contamination by mycotoxins and plant secondary metabolites is a potential issue, these systems have several advantages over either prokaryotic or animal cell expression system [1]. For example, plant cellderived transgenic proteins are likely to be safer for human use than those derived from mammalian cells, since plant cell contaminants and viruses are not pathogenic to humans. A plant cell system is able to carry out the post translational modiWcations whereas prokaryotic system
*
Corresponding author. Fax: +82 63 270 4334. E-mail address: [email protected] (M.-S. Yang).
1046-5928/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.09.028
cannot. In addition, sexual crossing can generate multiple transgenic plants. Additionally, plant cells are generally inexpensive to grow on a large scale, and their production is not limited to fermentor capabilities. Furthermore, it is much easier and more economical to purify secreted foreign proteins from plant cell media than from complex mammalian cell media. Therefore, plant cell culture systems may be the most suitable means of producing small-to-medium quantities of high-priced, high purity, specialty recombinant proteins. Human granulocyte-colony stimulating factor (hGCSF)1 was a member of the long chain family of cytokines, Wrst reported by Nagata et al. [2]. The mature hG-CSF is a 19.6 kDa glycoprotein with 174 amino acids and is produced mainly by monocytes and macrophages upon activation by endotoxin [3]. G-CSF plays a critical role in the
1 Abbreviations used: hG-CSF, human granulocyte-colony stimulating factor; HPT, hygromycin phosphotransferase.
S.-Y. Hong et al. / Protein Expression and PuriWcation 47 (2006) 68–73
process of hematopoiesis, regulating the proliferation, diVerentiation, and survival of neutrophils and neutrophilic progenitor cells [4]. G-CSF has been expressed in various foreign hosts such as Escherichia coli [5], yeast [6], and mammalian cells [7] and is now employed to treat cancer patients undergoing chemotherapy to alleviate the depression of white blood cell levels produced by cytotoxic therapeutic agents. We previously produced hG-CSF using transformed tobacco cells in suspension cultures, and demonstrated that it was secreted into the culture medium in a bioactive form [8]. The amount of hG-CSF present in the culture medium increased over time to a maximum of 105 g/L at day 9 after incubation and dropped quickly. To overcome this problem, we used the rice amylase expression system Ramy3D [9] to manufacture recombinant hG-CSF in rice cell suspension culture, and found that the yield of hG-CSF was signiWcantly higher than that obtained in our previous transgenic tobacco expression system. Materials and methods Expression vector construction The hG-CSF cDNA was synthesized using RT-PCR technique with poly(A)+ mRNA from TPA stimulated THP-1 cell line. A pair of primers (5⬘-AAA GTA CTC CTC TGG GTC CTG CTA GCT C-3⬘ and 5⬘-TTG GTA CCC TTG GCT CAG GGC TGG-3⬘) were designed to generate the 555 bp of PCR fragment containing an open reading frame for hG-CSF lacking its signal peptide [2]. The PCR product was cloned into pUC18 vector (TAKARA, Japan) to generate the pMYO83. The DNA fragment containing the Ramy3D promoter and signal
69
peptide coding sequence [9] was ampliWed with primers 5⬘GAG CAT GCA CCA CCT GTG CTA GCT ACT CCA CTG-3⬘ and 5⬘-AAC TGC AGG CTT GAC CCG AGT TAC AGG TC-3⬘. The PCR product was digested with SphI and PstI, and subcloned into the pUC18 multiple cloning site. The resultant plasmid, pMYO83, was digested with ScaI and KpnI to excise the gene for hG-CSF, which was subcloned into the PstI (blunted with T4 DNA polymerase) and KpnI site of pMYN27. The resulting plasmid, pMYO85, was digested with HindIII and EcoRI, and a DNA fragment containing hG-CSF with the Ramy3D promoter and signal peptide coding sequence was inserted into the binary vector (Fig. 1). Transformation and screening of transgenic rice calli Rice calli were prepared and transformed using the particle bombardment mediated transformation previously described [10]. After bombardment with expression vectors of hG-CSF gene, callus were transferred to selection medium N6 [11] supplemented with 2,4-dichlorophenoxyacetic acid (2 mg/L), sucrose (30 g/L), proline (0.5 g/ L), glutamine (0.5 g/L), casein enzymatic hydrolysate (0.3 g/L), and hygromycin B (35 mg/L) every 2–3 weeks. Calli resistant to hygromycin B were grown in N6 media minus sucrose (N6 ¡ S) for 7 days and then analyzed for hG-CSF in transgenic calli by ELISA. Calli were then ranked according to the hG-CSF expression level. Culturing on the same media for 2 months, with subculturing every 15 days, further established the suspension culture with selected calli. A single small healthy, oV-white, compact, and fast growing callus with doubling time about 7 days from each transformed line was grown on N6 medium for 10–15 days.
Fig. 1. Schematic diagram of the gene construct utilized in this study. Transferred-DNA (T-DNA) of the Wnal plasmid is shown. RB, T-DNA right border; 3⬘ UTR, 3⬘ untranslated region of rice -amlyase gene; du35S, CaMV35S promoter with a duplicated enhancer region; HPT, hygromycin phosphotransferase; Tnos, terminator of nopaline synthase; and LB, T-DNA left border.
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Northern and Western blot analyses For Northern blot analysis, total RNA was isolated from suspension cells grown in N6 + S and N6 ¡ S liquid media by using the RNeasy plant total RNA mini kit (Qiagen, CA, USA). Northern blot analysis was conducted according to the procedure of Sambrook et al. [12]. The blots for Northern blot analysis were hybridized with [32 P]-labeled random-primed DNA using about 555 bp ScaI and PstI fragment from pMYO83 that included hG-CSF gene. For Western blot analysis, samples were electroblotted onto Hybond C Extra membrane (Amersham–Pharmacia Biotechnology, UK) after SDS–PAGE. Rat monoclonal anti human G-CSF antibody (Pharmingen, CA) at 1:2000 and HRP-conjugated goat anti-rat IgG (Sigma, MO) at 1:7000 were used as primary and secondary antibodies, respectively. Measurement of quantitative analyses and biological activity of hG-CSF After production of recombinant hG-CSF was induced incubating transgenic rice cells in N6 ¡ S liquid medium, the suspension culture was centrifuged at 12,000g for 10 min. One millilitre sample of the resulting culture supernatant was dialyzed against PBS overnight at 4 °C, and used to measure the quantitative analyses and biological activity of hG-CSF. The concentration of rhG-CSF in the cultured media was determined by hG-CSF speciWc ELISA kit (Endogene, Woburn, MA) according to the procedure provided by manufacturer. To determine the biological activity of hG-CSF produced by rice cell suspension culture, minimal level of the sample for the induced proliferation of hG-CSF required AML-193 cells was measured [13]. BrieXy, growth-factor-starved 1 £ 105 cells were suspended in 100 L of RPMI 1640 medium (Sigma, MO) supplemented with 5% FBS (HyClone Laboratories, Logan, UT, USA). The 25 ng of hG-CSF produced from O85-4 cell line and E. coli derived hG-CSF were used for biological activity analysis. After 27 h incubation, 1 Ci of [methyl-3H]thymidine (Amersham Lifescience, NJ, USA) was added into each well and incubated for an additional 32 h. The cells were harvested with cell harvester (Inotech, Switzerland) and the tritium content was measured with a liquid scintillation counter (Packard, USA). In these experiments, a recombinant E. coli-derived human G-CSF, which was purchased from Pharmingen (San Diego, CA, USA), was used as a standard.
DNA sequence analysis using the dideoxynucleotide chain termination method. The structure of the hG-CSF expression vector pMYO85, which contains the hygromycin phosphotransferase (HPT) gene as a selection marker. The hG-CSF gene was placed under the control of the Ramy3D promoter, expression can be induced by removing the sucrose from the culture (Fig. 1). Rice calli were transformed with the pMYO85 plasmid by particle bombardment. Four weeks after bombardment transformation, hygromycin resistant rice callus colonies were generated. Preliminary screening of 27 putative transgenic cells for detection of hG-CSF expression was conducted by Western blot analysis. To conWrm the integration of hG-CSF in the transgenic rice cell lines, genomic DNA PCR analysis was performed with primers designed to amplify the hG-CSF gene (data not shown). Expression of recombinant hG-CSF gene and protein in transgenic rice suspension cells ELISA was conducted to initially screen hG-CSF protein expression in the conditioned media and cell extracts of transgenic cell suspension. As shown in Fig. 2, hG-CSF levels in the transgenic callus lines, which were measured at day 7 after transfer to sucrose-free plate, varied widely. Line O85-4 produced the highest level of recombinant hGCSF (185 g/g cells). This resulted in identiWcation of four ‘high-producer’ lines (O85-2, -4, -5, -7), which were selected for further analysis. Transcripts of recombinant hG-CSF gene expression in these lines were detected by Northern blot analysis. Similarly hG-CSF transcript signals were detected in transgenic cell lines under sucrose-starvation conditions (Fig. 3). The hG-CSF mRNA was not detected in wild-type cell line. As shown in Fig. 4A, levels of hG-CSF mRNA in O85-4 increased until day 7 after induction and then decreased slowly. A little hG-CSF mRNA was expressed by day 7 in sucrose medium. There was little sucrose in culture medium after day 7 in growth phase due to consumption by rice cells,
Results IdentiWcation and characterization of transgenic rice suspension cell lines The cDNA clone of hG-CSF was synthesized using the RT-PCR technique from TPA stimulated THP-1 cell line. The nucleotide sequence of hG-CSF was conWrmed by
Fig. 2. Screening of transformed callus lines expressing high levels of hGCSF. The amount of hG-CSF was determined by ELISA as described in Materials and methods. Lane NC is negative control which used suspension cells transformed with vector only. Lanes 1–8 represent the transgenic rice cell lines expressing the hG-CSF selected from the previous genomic DNA PCR analysis (data not shown).
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band of »20 kDa. The molecular weight of the hG-CSF was similar to that of the E. coli-derived hG-CSF. The hGCSF was not detectable in wild-type cell lines according to SDS–PAGE or Western blot analysis. QuantiWcation and functional assay for recombinant hG-CSF Fig. 3. Northern blot analysis to determine expression of hG-CSF gene during rice suspension cultures without sucrose. Each lane was loaded with 10 g total RNA from non-transgenic and transgenic rice cells. Loading standards were indicated by ethidium bromide stained-rRNA in lower panel. Lane NC is negative control which used suspension cells transformed with vector only. Lanes 1–4 represent the transgenic rice cell lines expressing the hG-CSF selected from the previous ELISA data.
Fig. 4. (A) Northern blot analysis to determine the temporal expression pattern of the hG-CSF gene during plant cell suspension culture. NC is negative control which used suspension cells transformed with vector only. S+ and S¡ indicate media with and without sucrose, respectively. Loading standards are indicated by ethidium bromide-stained rRNA in the lower panel. (B) SDS–PAGE, and (C) Western blot analysis of hGCSF in culture medium. Lanes M, PC, and NC denote prestained molecular weight standards, positive control hG-CSF derived from E. coli, and negative control culture medium from suspension cells transformed with the parent vector only, respectively. Lane 1 represents the total secreted protein obtained from the culture supernatant at day 7 in the N6 + S media. Lanes 2–5 indicate the total secreted protein obtained from the culture supernatant at days 1, 5, 11, and 15 in the N6 ¡ S media, respectively.
which induced the Ramy3D expression system. Levels of recombinant hG-CSF protein accumulated in culture medium were determined by Western blot analysis (Fig. 4C). In Fig. 4C, there were faint bands in lanes 1 and 2, which corresponded to day 7 in the growth phase and day 1 in the induction phase, respectively. The non-glycosylated E. coliderived recombinant hG-CSF appeared as a band at »20 kDa on the Western blot analysis. The culture medium from the transgenic rice cells produced hG-CSF protein
For the amount of hG-CSF in culture medium, results of time course experiments following total secreted protein and recombinant hG-CSF production are depicted in Fig. 5. ELISA was conducted for quantifying the secreted hG-CSF proteins in the suspension cells. The level of recombinant protein increased sharply in the production phase to a maximum of 2.5 mg/L at day 13. Recombinant hG-CSF comprised about 0.7% of the total secreted protein (Fig. 5). The biological activity of recombinant hG-CSF produced in transgenic rice cells was measured with E. coliderived hG-CSF, on AML-193 cells (Fig. 6). The culture
Fig. 5. Time course of hG-CSF and total secreted protein accumulated in culture medium of suspension cultures as determined by ELISA and Bradford assay, respectively. Rice cells in suspension culture were propagated for 9 days in N6 + S media, transferred to N6 ¡ S media, and cultured for the lengths of time (days) indicated.
Fig. 6. SpeciWc activity of recombinant hG-CSF produced from transgenic rice suspension cells. The 25 ng/ml of plant derived hG-CSF (phG-CSF) and E. coli-derived hG-CSF (PC) were used for biological activity analysis. NC is negative control which used culture medium of suspension cells transformed with vector only.
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system is a suitable system for production of valuable recombinant proteins combined with the advantages of plant cell culture.
medium from rice cells transformed with the parent vector only was used as a negative control, it did not support the growth of hG-CSF required AML-193 cells. The recombinant hG-CSF produced from the O85-4 cell line was eVectively dependent on the proliferation of AML-193 cells. The biological activity of the recombinant hG-CSF tested here matched completely with the data of ELISA, which demonstrated that it was correctly folded and functional. The speciWc activity of recombinant hG-CSF produced from transgenic rice cell suspension culture was similar to that exhibited by the commercial E. coli-derived hG-CSF.
This research was supported by a grant (Next Generation New Technology Development Project) from MOCIE, Republic of Korea. Dr. Soo-Ho Kim was supported by the post-doctoral program of Chonbuk National University, Republic of Korea.
Discussion
References
In this study, we have characterized the suitability of genetically engineered rice cell suspension culture system for the expression of recombinant human G-CSF. Human G-CSF have previously been expressed and secreted into culture media in a biologically active form [8]. The amount of hG-CSF produced and secreted by cultured transgenic tobacco suspension was about 105 g/L in 9 days after the initiation of the cell suspension culture. After 9 days, however, the rapid decrease in the amount of recombinant protein in the culture medium was observed. Consistent with our Wndings, previous reports suggests that, when present in plant cell culture medium, the stability and accumulation of recombinant proteins may decrease [14,15]. For this study, improving accumulation of target proteins in cell culture media by the use of protein stability agents such as PVP, gelatin and mannitol [16] has been explored. Hence, we opted to use a sucrose-starvation inducible, -amylase 3D promoter system. When rice amylase genes are expressed in the germinating seed, the Ramy3D was expressed in rice culture cells during sugar starvation. Terashima et al. [17] and Stoger et al. [18] demonstrated observable eVects in expression of pharmaceutical proteins in plant bioreactor systems, using Ramy3D promoter. We have shown previously that under the control of Ramy3D signal peptide, recombinant human granulocyte–macrophage colony stimulating factor were secreted into the cell culture medium [19]. We also found that protease activity in the culture medium upon induction of the Ramy3D promoter by sucrose starvation was very low. By exploiting this expression system, we increased the production of hG-CSF compared with our previous expression system, which utilized tobacco cell suspension cultures and the CaMV 35S promoter. The amount of recombinant protein in the medium increased until 13 days after induction, after which it dropped quickly. The maximum amount of hG-CSF was obtained in the rice cell culture medium with the Ramy3D expression systems. We suggest, based on this study that the current transgenic rice cell suspension culture system, especially Ramy3D, can be further developed as an experimental bio-reactor system for evaluating and upgrading the production of hG-CSF proteins in plant system. We therefore believe that the Ramy 3D expression
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Acknowledgment
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