Abstract
Peptide and RNA epitope tags as tools for routine analysis of transgene expression and protein accumulation in transformed plant cell cultures was evaluated using three genes that encode very structurally and functionally different proteins. A T7 peptide was introduced at the amino- and carboxyl-termini of phosphinothricin-N-acetyl transferase and avidin and at the carboxyl-terminus of galactose oxidase. An RNA sequence that forms a higher order structure that is recognized by antibodies raised against the FLAG peptide was separately introduced into the 3′ nontranslated region of these genes. Constructs were introduced into maize cell cultures using particle bombardment and transgene expression, protein accumulation, protein function and presence of the tags in RNA and/or protein as appropriate were evaluated in up to approximately 25 culture lines per construct. Results indicate that, while there will likely always be a need for some empirical evaluation of any tag-protein combination, introduction of the peptide tag at the amino-terminus was generally more successful than was incorporation at the carboxyl-terminus. RNA tags show promise for this purpose, but routine application will require development of a very sensitive immunoassay.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Armstrong CL (1994) In: Freeling M and Walbot V (eds). The Maize Handbook. (pp. 663–670) Springer, New York.
Bevan M, Barnes WM and Chilton MD (1983) Structure and transcription of the nopaline synthase gene region of T-DNA. Nucl Acids Res 11: 369–385.
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248–254.
Brusa R (1999) Genetically modified mice in neuropharmacology. Pharmacol Res 39: 405–419.
Chomczynski P and Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochem 162: 156–159.
Christensen AH, Sharrock RA and Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplast by electroporation. Plant Mol Biol 18: 675–689.
De Blok M, Botterman J, Vandewiele M, Dockx J, Thoen C, Gossele V, Movva NR et al. (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J 6: 2513–2518.
Deutscher SL, Keene JD (1988) A sequence-specific conformational epitope on U1 RNA is recognized by a unique autoantibody. Proc Natl Acad Sci USA 85: 3299–3303.
Droge W, Broer I and Puhler A (1992) Transgenic plants containing the phosphinothricin-N-acetyltransferase gene metabolize the herbicide L-phosphinothricin (glufosinate) differently from untransformed plants. Planta 187: 142–151.
Ferrando A, Farras R, Jasik J, Schell J and Koncz C (2000) Introntagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells. The Plant J 22: 553–560.
Gope ML, Keinänen RA, Kristo PA, Conneely OM, Beattie WG, Zarucki-Schulz T et al. (1987) Molecular cloning of the chicken avidin cDNA. Nucl Acids Res 15: 3595–3606.
Ho SN, Hunt HD, Horton RM, Pullen JK and Pease LR (1989) Sitedirected mutagenesis by overlap extension using the polymerase chain reaction. Gene 77: 51–59.
Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M et al. (1997) Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol Breeding 3: 291–306.
Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Cerretti DP et al. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Biotechnology 6: 1204–1210.
Jarvik JW and Telmer CA (1998) Epitope tagging. Ann Rev Genetics 32: 601–618.
Keene JD, Kenan DJ and Tsai DE (1992) Method for deriving epitopes PCT/US93/08210.
Kreis TE (1986). Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell Surface. EMBO J 5: 931–941.
Kolodziej P and Young RA (1991). Epitope tagging and protein surveillance. Methods in Enzymol 194: 508–519.
Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L et al. (1993) Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 11: 194–200.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Ma JKC, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, Hein MB and Lehner T (1998) Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nature-Medicine 4: 601–606.
McPherson MJ, Ogel ZB, Stevens C, Yadav KDS, Keen JN and Knowles PF (1992) Galactose oxidase of Dactylium dendroides. The J Biol Chem 267: 8146–8152.
Register JC III, Peterson DJ, Bell PJ, Bullock WP, Evans IJ, Frame B et al. (1994) Structure and function of selectable and nonselectable transgenes in maize after introduction by particle bombardment. Plant Mol Biol 25: 951–961.
Towbin H, Staehelin T and Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nat Acad Sci USA 76: 4350–4354.
Tomes DT, Ross MC and Songstad DD (1995) Direct DNA transfer into intact plant cells via microprojectile bombardment. In: Gamborg OL and Philips GC (eds), Plant Cell Tissue and Organ Culture: Fundamental Methods. (pp. 197–213) Springer, Berlin.
Tsai DE, Kenan DJ and Keene JD (1992) In vitro selection of an RNA epitope immunologically cross-reactive with a peptide. Proc Natl Acad Sci USA 89, 8864–8868.
Van Cott KE, Lubon H, Russell CG, Butler SP, Gwazdauskas FC, Knight J et al. (1997) Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C. Transgenic Res 6: 203–212.
Van Venrooij W J, Hoet R, Castrop J, Hageman B, Mattaj IW and van de Putte LB (1990) Anti-(U1) small nuclear RNA antibodies in anti-small nuclear ribonucleoprotein sera from patients with connective tissue diseases. J Clin Invest 86: 2154–2160.
Wohlleben W, Arnold W, Broer, I, Hillemann, D, Strauch E and Puhler A (1988) A nucleotide sequence of the phosphinothricin N-acetyltransferase gene form Streptomyces viridochromogenes Tu494 and its expression in Nicotiana tabacum. Gene 70: 25–37.
Zhou M, Diwu Z, Panchuk-Voloshina N and Haugland R (1997) A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 253: 162–168.
Author information
Authors and Affiliations
Additional information
Both of these authors contributed equally to this work and should be recognized as first authors.
Rights and permissions
About this article
Cite this article
Alarcon, C.M., Umthun, A.R. & Register, J.C. Use of epitope tags for routine analysis of transgene expression. Transgenic Res 10, 183–192 (2001). https://doi.org/10.1023/A:1016633208182
Issue Date:
DOI: https://doi.org/10.1023/A:1016633208182