Abstract
The gene encoding neomycin phosphotransferase II (NPTII) has been used routinely as a selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the same coding sequence used for plant transformation was introduced into Escherichia coli to produce gram quantities of this protein. A unique, simple, rapid and efficient purification method was developed to purify thirty grams of NPTII protein. The microbially produced NPTII was shown to be chemically and functionally equivalent to the NPTII protein expressed in and purified from genetically engineered cotton seed, potato tubers and tomato fruit. Microbially produced and plant produced NPTII proteins have comparable molecular weights, immuno-reactivities, epitope structures, amino terminal amino acid sequences, biological activities and both lack glycosylation. Demonstrating the equivalence of NPTII protein from these sources establishes the validity of using the microbially produced NPTII to assess the safety of the NPTII protein produced in genetically engineered crops.
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References
Gasser, C.S. and Fraley, R.T. 1989. Genetically engineering plants for crop improvement. Science 244: 1293–1299.
Redenbaugh, K., Hiatt, W., Martineau, B., Kramer, M., Sheehy, R., Sanders, R., Houck, C. and Emlay, D. 1992. Safety Assessment of Genetically Engineered Fruits and Vegetables: A Case Study of the Flavr Savr® Tomato, CRC Press, Boca Raton, FL.
Animal and Plant Health Inspection Service. 1992. Notice of proposed interpretive ruling in connection with the Upjohn Company petition for determination of regulatory status of ZW020 virus resistant squash. Fed. Reg. 57: 40632–40633.
Animal and Plant Health Inspection Service. 1992. Proposed interpretive ruling in connection with Calgene, Inc. petition for determination of regulatory status of Flavr Savr® Tomato . Fed. Reg. 57: 31170.
Animal and Plant Health Inspection Service. 1993. Genetically engineered organisms and products; notification procedures for the introduction of certain regulated articles; and petition for nonregulated status; final rule. Fed Reg. 58: 1704–17059.
Flavell, R.B., Dart, E., Fuchs, R.L. and Fraley, R.T. 1992. Selectable marker genes: safe for plants? Bio/Technology 10: 141–144.
Nap, J.P., Bijvoet, J. and Stikema, W.J. 1992. Biosafety of kanamycin-resistant transgenic plants: an overview. Transgenic Crops 1: 239–249.
Fuchs, R.L., Ream, J.E., Hammond, B.G., Naylor, M.W. and Berberich, S.A. 1993. Safety of the neomycin phosphotransferase II (NPTII) protein. Bio/Technology 11: This issue.
Perlak, F.J., Deaton, R.W., Armstrong, T.A., Fuchs, R.L., Sims, S.R., Greenplate, J.T. and Fischhoff, D.A. 1990. Insect resistant cotton plants. Bio/Technology 8: 939–943.
Perlak, F., Stone, T.B., Muskopf, Y.M., Petersen, L.J., Parker, G.B., McPherson, S.A., Wyman, J., Love, S., Beaver, D., Reed, G. and Fischhoff, D. 1993. Genetically improved potatoes—protection from damage by Colorado potato beetles. Plant Mol. Biol. 22: 313–321.
Klee, H.J., Harford, M.B., Kretzmer, K.A., Barry, G.F. and Kishore, G.M. 1991. Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. The Plant Cell 3: 1187–1193.
Rogan, G.J., Ream, J.E., Berberich, S.A. and Fuchs, R.L. 1992. Enzyme-linked immunosorbent assay for quantitation of neomycin phosphotransferase II in genetically modified cotton tissue extracts. J. Agric. Food Chem. 40: 1453–1458.
Olins, P.O., Rangwala, S.H., Devine, C.S. and Kauka, K.S. 1988. The T7 phage gene 10 leader, a ribosome binding site that dramatically enhances the expression of foreign genes in E. coli. Gene 73: 227–235.
Beck, E., Ludwig, G., Auerswald, E., Reiss, B. and Schaller, H. 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 9: 327–336.
Goldman, P.R. and Northrop, D.B. 1976. Purification and spectrophotometric assay of neomycin phosphotransferase II. Biochem. Biophys. Res. Comm. 69: 230–236.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248–254.
Michowitz, M., Leibovici, J. and Wolman, M. 1979. Protective effect of native levan on endotoxin in mice and rats. Experientia 35: 804–805.
Wood, D.C., Vu, L.V., Kimack, N.M., Rogan, G.J. and Nickson, T.E. 1993. Purification and N-terminal sequence of neomycin phosphotransferase II (NPTII) from genetically modified cotton seed (Gossypium hirsutum). Protein Expression Purification. In press.
Rodbard, D., Munson, P. and DeLean, A. 1978. Improved curve fitting, parallelism testing, characterization of sensitivity and specificity, validation and optimization for radioligand assays, p. 469–504. In: Radioimmunoassay and Related Procedures in Medicine, Vol 1. IAEA, Vienna.
Slater, R. 1988. The expression of foreign DNA in Escherichia coli, p. 47–67. In: Molecular Biology and Biotechnology. J. M. Walker and E. B. Gingold (Eds.). Burlington House, London.
Taiz, L. and Zeiger, E. 1991. Plant Physiology. The Benjamin/Cummings Publishing Co., Inc., Redwood City, CA.
Food and Drug Administration. Department of Health and Human Services. 1993. Direct food substances affirmed as generally recognized as safe; chymosin enzyme preparation derived from Aspergillus niger van Tieghem variety awamori (Nakazawa) Al-Musaliam. Fed. Reg. 58: 27197–27203.
Obukowicz, M.G., Turner, M.A., Wong, E.Y. and Tacon, W.C. 1988. Secretion and export of IGF-1 in Escherichia coli strain JM101. Mol. Gen Genet. 215: 19–25.
Moore, S. and Stein, W.H. 1963. Chromatographic determination of amino acids by the use of automatic recording equipment. Methods in Enzymol 6: 819–831.
Hunkapiller, M.W. 1983. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods in Enzymol. 91: 399–413.
Perlin, M.H., McCarty, S.C. and Greer, J.P. 1988. Coupled spectroflourometric assay for aminoglycoside phosphotransferases. Anal. Biochem. 171: 145–149
McDonald, R.E., Clark, R.D., Smith, W.A. and Hinchee, M.A. 1987. Simplified method for the detection of neomycin phosphotransferase activity in transformed tissue. Plant Mol. Biol. Rep. 5: 380–386.
Laemmli, U.K. 1970. Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 227: 680–685.
Neuhoff, V., Arold, N., Taub, D. and Ehrhardt, W. 1988. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie brilliant blue G-250 and R-250. Electrophoresis 9: 255–260.
Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262: 10035–10038.
Domingo, A. and Marco, R. 1988. Visualization under ultraviolet light enhances 100-fold the sensitivity of peroxidase-stained blots. Anal. Biochem 186: 176–181.
Mitchell, J. and Smith, D.M. 1980. Aquametry: Part I and II, John Wiley & Sons, New York.
Small, H., Stevens, T. and Bauman, W. 1975. Novel ion exchange chromatographic method using conductimetric detection. Anal. Chem. 47: 1801–1809.
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Fuchs, R., Heeren, R., Gustafson, M. et al. Purification and Characterization of Microbially Expressed Neomycin Phosphotransferase II (NPTII) Protein and its Equivalence to the Plant Expressed Protein. Nat Biotechnol 11, 1537–1542 (1993). https://doi.org/10.1038/nbt1293-1537
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DOI: https://doi.org/10.1038/nbt1293-1537
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