Abstract
Recombinant proteins have been previously synthesized in a transgenic rice cell suspension culture system with the rice amylase 3D promoter, which can be induced via sugar starvation. However, the secreted recombinant proteins have been shown to be rapidly decreased as the result of proteolytic degradation occurring during prolonged incubation. The secreted proteases were identified via two-dimensional electrophoresis (2-DE) and ESI/Q-TOF mass spectrometry analyses. The internal amino acid sequences of 8 of 37 spots corresponded to cysteine proteinase (CysP), which is encoded for by Rep1 and EP3A. This result shows that CysP is a major secreted protease in rice cell suspension cultures following induction via sugar starvation. Intron-containing self-complementary hairpin RNA (ihpRNA)-mediated post-transcriptional gene silencing (PTGS) was applied to suppress the expression of CysP in rice cell suspension cultures. The reduction of rice CysP mRNA and the detection of siRNA specific to CysP, an initiator of RNAi, were verified via Northern blot analysis and RNase protection assays, respectively, thereby indicating that PTGS operated successfully in this system. The analysis of total secreted protease and CysP activities evidenced lower activity than was observed with the wild-type. Furthermore, suspension cultures of rice cells transformed with both hGM-CSF and the gene expressing the ihpRNA of CysP evidenced a reduction in total protease and CysP activities, and an up to 1.9-fold improvement in hGM-CSF production as compared to that observed in a rice cell line expressing hGM-CSF only. These results demonstrate the feasibility of the suppression of CysP via RNA interference to reduce protease activity and to increase target protein accumulation in rice cell suspension cultures.
Similar content being viewed by others
References
Azevedo C, Sadanandom A, Kitagawa K et al (2002) The RAR1 interactor SGT1, an essential component of R gene-triggered disease resistance. Science 295:2073–2076. doi:10.1126/science.1067554
Balland A, Krasts DA, Hoch KL et al (1998) Characterization of the microheterogeneities of PIXY321, a genetically engineered granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein expressed in yeast. Eur J Biochem 251:812–820. doi:10.1046/j.1432-1327.1998.2510812.x
Barrett AJ, Kirschke H (1981) Cathepsin B, cathepsin H and cathepsin L. Methods Enzymol 80:535–561
Bernstein E, Caudy AA, Hammond SM et al (2001) Role for a bidentate ribonuclease in the initiation step of RNA interferences. Nature 409:363–366. doi:10.1038/35053110
Chan MT, Yu SM (1998a) The 3′ untranslated region of a rice α-amylase gene mediates sugar-dependent abundance of mRNA. Plant J 15:685–695. doi:10.1046/j.1365-313x.1998.00244.x
Chan MT, Yu SM (1998b) The 3′ untranslated region of a rice α-amylase gene functions as a sugar-dependent mRNA stability determinant. Proc Natl Acad Sci USA 95:6543–6547. doi:10.1073/pnas.95.11.6543
Chen LL, Marmey P, Taylor NJ et al (1998) Expression and inheritance of multiple transgenes in rice plants. Nat Biotechnol 16:1060–1064. doi:10.1038/3455
Chiou CJ, Wu MC (1990) Expression of human granulocyte-macrophage colony-stimulating factor gene in insect cells by a baculovirus vector. FEBS Lett 259:249–253. doi:10.1016/0014-5793(90)80020-J
Chu CC, Wang CC, Sun CS (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci Sin 18:659–668
Chuang CF, Meyerowitz EM (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 97:4985–4990. doi:10.1073/pnas.060034297
Dale DC, Liles WC, Summer WR (1995) Granulocyte-colony-stimulating factor (G-CSF): role in relationships in infectious diseases. J Infect Dis 172:1061–1075
Fischer R, Liao YC, Hoffman K et al (1999) Molecular farming of recombinant antibodies in plants. Biol Chem 380:825–839. doi:10.1515/BC.1999.102
Guo HS, Fei JF, Xie Q et al (2003) A chemical-regulated inducible RNAi system in plants. Plant J 34:383–392. doi:10.1046/j.1365-313X.2003.01723.x
Hajdukiewicz P, Svab Z, Maliga P (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994. doi:10.1007/BF00014672
Hammond SM, Bernstein E, Beach D et al (2000) An RNA-directed nuclease mediates posttranscriptional gene silencing in Drosophila cells. Nature 404:293–296. doi:10.1038/35005107
Hayama R, Yokoi S, Tamaki S et al (2003) Adaptation of photoperiodic control pathways produces short-day flowering in rice. Nature 422:719–722. doi:10.1038/nature01549
Hellwig S, Drossard J, Twyman RM et al (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422. doi:10.1038/nbt1027
Ho SL, Tong WF, Yu SM (2000) Multiple mode regulation of a cysteine proteinase gene expression in rice. Plant Physiol 122:57–66. doi:10.1104/pp.122.1.57
Kim MJ, Kwon TH, Jang YS et al (2000) Expression of murine GM-CSF in recombinant Aspergillus niger. J Microbiol Biotechnol 10:287–292. doi:10.1159/000056641
Kim TG, Kim HM, Lee HJ et al (2007) Reduced protease activity in transformed rice cell suspension cultures expressing a proteinase inhibitor. Protein Expr Purif 53:270–274. doi:10.1016/j.pep.2007.01.005
LaCount W, An G, Lee JM (1997) The effect of polyvinylpyrrolidone (PVP) on the heavy chain monoclonal antibody production from plant suspension cultures. Biotechnol Lett 19:93–96. doi:10.1023/A:1018383524389
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0
Lee F, Yokota T, Otsuka T et al (1985) Isolation of cDNA for a human granulocyte-macrophage colony stimulation factor by functional expression in mammalian cells. Proc Natl Acad Sci USA 82:4360–4364. doi:10.1073/pnas.82.13.4360
Lee JS, Choi SJ, Kang HS et al (1997) Establishment of a transgenic tobacco cell suspension culture system for producing murine granulocyte-macrophage colony stimulating factor. Mol Cells 7:783–787
Lee JH, Kim NS, Kwon TH et al (2002) Increased production of human granulocyte-macrophage colony stimulating factor (hGM-CSF) by the addition of stabilizing polymer in plant suspension cultures. J Biotechnol 96:205–211. doi:10.1016/S0168-1656(02)00044-5
Lee SY, Cho JM, Kim DI (2003) Effects of bacitracin on hGM-CSF production in suspension cultures of transgenic Nicotiana tabacum cells. Enzyme Microb Technol 33:353–357. doi:10.1016/S0141-0229(03)00145-5
Libby RT, Braedt G, Kronheim SR et al (1987) Expression and purification of native human granulocyte-macrophage colony-stimulating factor from an Escherichia coli secretion vector. DNA 6:221–229
Ma JKC, Lehner T, Stabila P et al (1994) Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur J Immunol 24:131–138. doi:10.1002/eji.1830240120
Martinez J, Patkaniowska A, Urlaub H (2002) Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110:563–574. doi:10.1016/S0092-8674(02)00908-X
Metcalf D (1985) The granulocyte-macrophage colony-stimulating factors. Science 229:16–22. doi:10.1126/science.2990035
Metcalf D (1991) Control of granulocyte and macrophages, molecular, cellular, and clinical aspects. Science 254:532–533. doi:10.1126/science.1948028
Moritoh S, Miki D, Akiyama M et al (2005) RNAi-mediated silencing of OsGEN-L (OsGEN-like), a new member of the RAD2/XPG nuclease family, causes male sterility by defect of microspore development in rice. Plant Cell Physiol 46:699–715. doi:10.1093/pcp/pci090
Nykanen M, Saarelainen R, Raudaskoski M et al (1997) Expression and secretion of barley cysteine endopeptidase B and cellobiohydrolase I in Trichoderma reesei. Appl Microbiol Biotechnol 63:4929–4937
O’Farrell PH (1997) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
Outchkourov N, Rogelj B, Strukelj B et al (2003) Expression of sea anemone equistatin in potato. Effects of plant protease on heterologus protein production. Plant Physiol 133:379–390. doi:10.1104/pp.102.017293
Russell DA (1999) Feasibility of antibody production in plant for human therapeutic use. In: Hammond J, McGarvey P, Yusibov V (eds) Plant biotechnology, new products and application. Springer, Berlin, pp 119–138
Russell D, Spatola L, Dian T et al (2005) Host limits to accurate human growth hormone production in multiple plant systems. Biotechnol Bioeng 89:775–782. doi:10.1002/bit.20366
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Saravanan RS, Rose JK (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4:2522–2532. doi:10.1002/pmic.200300789
Schiermeyer A, Schinkel H, Apel S et al (2005) Production of Desmodus rotundus salivary plasminogen activator α1 (DSPAα1) in tobacco is hampered by proteolysis. Biotechnol Bioeng 89:848–858. doi:10.1002/bit.20410
Sharp J, Doran P (1999) Effect of bacitracin on growth and monoclonal antibody production by tobacco hairy roots and cell suspension. Biotechnol Bioproc Eng 4:253–258
Sharp J, Doran P (2001) Characterization of monoclonal antibody fragments produced by plant cells. Biotechnol Bioeng 73:338–346. doi:10.1002/bit.1067
Shin YJ, Hong SY, Kwon TH et al (2003) High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnol Bioeng 82:778–783. doi:10.1002/bit.10635
Terashima M, Murai Y, Kawamura M et al (1999) Production of functional human α1-antitrypsin by plant cell culture. Appl Microbiol Biotechnol 52:516–523. doi:10.1007/s002530051554
Thompson JA, Abdullah R, Cocking EC (1986) Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose. Plant Sci 47:123–133. doi:10.1016/0168-9452(86)90059-2
Twyman RM, Stoger E, Schillberg S et al (2003) Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21:570–578. doi:10.1016/j.tibtech.2003.10.002
Waterhouse PM, Graham MW, Wang MB (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95:13959–13964. doi:10.1073/pnas.95.23.13959
Wongsamuth R, Doran P (1997) Production of monoclonal antibody by tobacco hairy roots. Biotechnol Bioeng 54:401–415. doi:10.1002/(SICI)1097-0290(19970605)54:5<401::AID-BIT1>3.0.CO;2-I
Zamore PD, Tuschl T, Sharp PA et al (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33. doi:10.1016/S0092-8674(00)80620-0
Acknowledgements
This work was supported by a grant from the Next Generation New Technology Development program of the Ministry of Commerce, Industry and Energy, and the Post-Doc. Program at Chonbuk National University (the second half term of 2006).
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Kim, NS., Kim, TG., Kim, OH. et al. Improvement of recombinant hGM-CSF production by suppression of cysteine proteinase gene expression using RNA interference in a transgenic rice culture. Plant Mol Biol 68, 263–275 (2008). https://doi.org/10.1007/s11103-008-9367-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-008-9367-8