Abstract
Several proteins are recalcitrant to expression in Escherichiacoli. To explore transgenic plants as an alternative expressionsystem, the gene encoding the potential herbicide target sedoheptulose-1,7-bisphosphatase (SBPase, EC 3.1.3.37) was expressed in transgenic tobacco(Nicotiana tabaccum) under the control of a duplicatedCaMV 35S RNA promoter. The active protein, a key enzyme in the Calvin cycle,accumulated to approximately 1.2% of total soluble protein. In order to purifyrecombinant SBPase, a sequence encoding six histidine residues was insertedC-terminally which allows a one step purification via Ni2+-NTAaffinity chromatography. N-terminal amino acid sequence analysis of the purifiedprotein confirmed processing of the transit peptide and revealed the previouslyunknown cleavage site. The transit peptide consists of 67 amino acids followedby the mature SBPase subunit of 342 amino acids including the C-terminalfusion. Purified SBPase was found to be enzymatically active after reduction with DTTand showed many biochemical properties of the native enzyme such as thedependence on Mg2+ and a pH optimum of 8.3. Subsequently, SBPaseproduced in transgenic tobacco was used in large-scale screening for thediscovery of novel herbicides.
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Ashton A.R. 1998. A simple procedure for purifiying the major chloroplast fructose-1,6-bisphosphatase from spinach (Spinacia oleracea) and characterization of its stimulation by sub-femtomolar mercuric ions. Arch Biochem Biophys 2: 207-224.
Berg D., Tietjen K., Wollweber D. and Hain R. 1999. From genes to targets: impact of functional genomics on herbicide discovery. Brighton Conf-Weeds 2: 491-500.
Bosch D., Smal J. and Krebbers E. 1994. A trout growth hormone is expressed, correctly folded and partially glycosylated in the leaves but not in the seeds of transgenic plants. Transgenic Res 3: 304-310.
Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72: 248-254.
Breazeale V.D., Buchanan B.B. and Wolosiuk R.A. 1978. Chloroplast sedoheptulose-1,7-bisphosphatase: Evidence for regulation by the ferredoxin/thioredoxin system. Z Naturforsch 33: 521-528.
Buchanan B.B. 1980. Role of light in the regulation of chloroplast enzymes. Ann Rev Plant Pysiol 31: 341-374.
Cadet F., Meunier J.-C. and Ferte N. 1987. Isolation and purification of chloroplastic spinach (Spinacia oleracea) sedoheptulose-1,7-bisphosphatase. Biochem J 241: 71-74.
Cadet F. and Meunier J.-C. 1988. pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea). Biochem J 253: 249-254.
Cadet F. and Meunier J.-C. 1988. Spinach (Spinacia oleracea) chloroplast sedoheptulose-1,7-bisphosphatase: Activation and deactivation, and immunological relationship to fructose-1,6-bisphosphatase. Biochem J 253: 243-248.
Corpet F. 1988. Multiple sequence alignment with hierarchial clustering. Nucl Acids Res 16: 10881-10890.
Dunford R.P., Durrant M.C., Catley M.A. and Dyer T.A. 1998. Location of the redox-active cysteines in chloroplast sedoheptulose-1,7-bisphosphatase indicates that its allosteric regulation is similar but not identical to that of fructose-1, 6-bisphosphatase. Photosynthesis Res 58: 221-230.
Fischer R., Drossard J., Commandeur U., Schillberg S. and Emans N. 1999. Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants. Biotechnol Appl Biochem 30: 101-108.
Franken E., Teuschel U. and Hain R. 1997. Recombinant proteins from transgenic plants. Curr Opin Biotech 8: 411-416.
Harrison E.P., Willingham N.M., Lloyd J.C. and Raines C.A. 1998. Reduced sedoheptulose-1,7-bisphosphatase levels in transgenic tobacco lead to decreased photosynthetic capacity and altered carbohydrate accumulation. Planta 204: 27-36.
von Heijne G., Steppuhn J. and Herrmann R.G. 1989. Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535-545.
Higo K., Saito Y. and Higo H. 1993. Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35S promoter in transgenic tobacco. Biosci Biotech Biochem 57: 1477-1481.
Horsch R.B., Fry J.E., Hoffman N.L., Rogers S.G. and Fraley R.T. 1985. A simple and general method for transferring genes into plants. Science 227: 1229-1231.
Koncz C. and Schell J. 1986. The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204: 383-396.
Kossmann J., Sonnewald U. and Willmitzer L. 1994. Reduction of the chloroplastic fructose-1, 6-bisphosphatase in transgenic potato plants impairs photosynthesis and plant growth. Plant J 6: 637-650.
Kozak M. 1984. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucl Acids Res 2: 857-872.
Kyhse-Andersen J. 1984. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Meth 10: 203-209.
Laemmli U.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277: 680-685.
Lanzetta P.A., Alvarez L.J., Reinach P.S. and Candia O.A. 1979. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100: 95-97.
Liege B., Shengwu M., Zhao D. and van Huystee R.B. 1998. Cationic peanut peroxidase: expression and characterization in transgenic tobacco and purification of the histidine-tagged protein. Plant Sci 136: 159-168.
Linsmaier E.M. and Skoog F. 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100-127.
Ma J. and Hein M. 1995. Plant antibodies for immunotherapy. Plant Physiol 109: 431-446.
Maliga P.S., Breznovis A. and Marton L. 1973. Streptomycin-resistant plants from callus culture of haploid tobacco. Nature New Biol 244: 29-39.
Martin W., Mustafa A.-Z., Henze K. and Schnarrenberger C. 1996. Higher-plant chloroplast and cytosolic fructose-1,6-bisphosphatase isoenzymes: origins via duplication rather than prokaryote-eucaryote divergence. Plant Mol Biol 32: 485-491.
Mason H. and Arntzen C.J. 1995. Transgenic plants as vaccine production systems. Trends Biotechnol 13: 388-392.
Mejare M., Lilius G. and Bülow L. 1998. Evaluation of genetically attached histidine affinity tails for purification of lactate dehydrogenase from transgenic tobacco. Plant Sci 134: 103-114.
Paul M.J., Knight J.S., Habash D., Parry M.A.J., Lawlor D.W., Barnes S.A. et al. 1995. Reduction in phosphoribulokinase activity by antisense RNA in transgenic tobacco: effect on CO2 assimilation and growth in low irradiance. Plant J 7: 535-542.
Poolman M.G., Fell D.A. and Thomas S. 2000. Modelling photosynthesis and its control. J Exp Bot 51: 319-328.
Price G.D., Evans J.R., von Caemmerer S., Yu J.-W. and Badger M.R. 1995. Specific reduction of chloroplast glyceraldehyde-3-phosphate dehydrogenase activity by antisense RNA reduces CO2 assimilation via a reduction in ribulose bisphosphate regeneration in transgenic tobacco plants. Planta 195: 369-378.
Raines C.A., Lloyd J.C., Willingham N.M., Potts S. and Dyer T. 1992. cDNA and gene sequences of wheat chloroplast sedoheptulose-1,7-bisphosphatase reveal homology with fructose-1,6-bisphosphatases. Eur J Biochem 205: 1053-1059.
Raines C.A., Lloyd J.C. and Dyer T.A. 1999. New insights into the structure and function of sedoheptulose-1,7-bisphosphatase; an important but neglected Calvin cycle enzyme. J Exp Bot 50: 1-8.
Sambrook J., Fritsch E.F. and Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.
Sijmons P.C., Dekker B.M.M., Schrammeijer B., Verwoerd T.C., van den Elzen P.J.M. and Hoekema A. 1990. Production of correctly processed human serum albumin in transgenic plants. Bio/Technology 8: 217-221.
Töpfer R., Matzeit V., Gronenborn B., Schell J. and Steinbiβ H.H. 1987. A set of plant expression vectors for transcriptional and translational fusions. Nucl Acids Res 14.
Verwoerd T.C., van Paridon P.A., van Ooyen A.J.J., van Lent J.W.M., Hoekema A. and Pen J. 1995. Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol 109: 1199-1205.
Voss A., Niersbach M., Hain R., Hirsch H.J., Liao Y.C., Kreuzaler F. et al. 1995. Reduced virus infectivity in N. tabaccum secreting a TMV-specific full-size antibody. Mol Breeding 1: 39-50.
Willingham N.M., Lloyd J.C. and Raines C.A. 1994. Molecular cloning of the Arabidopsis thaliana sedoheptulose-1, 7-bisphosphatase gene and expression studies in wheat and Arabidopsis thaliana. Plant Mol Biol 26: 1191-1200.
Wolosiuk R.A., Hertig C.M., Nishizawa A.N. and Buchanan B. 1982. Enzyme regulation in C4 Photosynthesis: Role of Ca2+ in thioredoxin-linked activation of sedoheptulose bisphosphatase from corn leaves. FEBS Lett 140: 31-35.
Woodrow I.E., Murphy D.J. and Latzko E. 1984. Regulation of stromal Sedoheptulose-1, 7-Bisphosphatase activity by pH and Mg2+ concentration. J Biol Chem 259: 3791-3795.
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Seuter, A., Busch, M. & Hain, R. Overexpression of the potential herbicide target sedoheptulose-1,7-bisphosphatase from Spinacia oleracea in transgenic tobacco. Molecular Breeding 9, 53–61 (2002). https://doi.org/10.1023/A:1019297521424
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DOI: https://doi.org/10.1023/A:1019297521424