Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin‐LR

We describe the engineering, regeneration, and characterization of transgenic tobacco plants expressing a recombinant antibody specific to microcystin‐LR (MC‐LR), the environmental toxin pollutant produced by species of cyanobacteria. The antibody was created by a genetic fusion of the antigen‐binding regions of the microcystin‐specific single‐chain antibody, 3A8, with constant regions from the murine IgG1κ, Guy's 13. IgG transgenes were controlled by a leader peptide that targets the transgene products to the secretory pathway and also allows for rhizosecretion. The antibody, extracted from the leaves or rhizosecreted into hydroponic medium by transgenic plants, was shown to have functional binding to MC‐LR. Antibody yields in transgenic plant leaves reached a maximum of 64 µg/g leaf fresh weight (0.6% total soluble protein), and the rate of antibody rhizosecretion reached a maximum of 5 µg/g root dry weight/24 h. Rhizosecreted antibody bound to MC‐LR in hydroponic medium, and transgenic plants grew more efficiently on medium containing MC‐LR compared to wild‐type controls. This proof of concept paves the way for applications to produce diagnostic antibodies to microcystin‐LR, remove it from the environment by phytoremediation, or enhance yields in crops exposed to MC‐LR.—Drake, P. M. W., Barbi, T., Drever, M. R., van Dolleweerd, C. J., Porter, A. J. R., Ma, J. K.‐C. Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin‐LR. FASEB J. 24, 882–890 (2010). www.fasebj.org

[1]  Y. Ueno,et al.  Detection and identification of microcystins in the drinking water of Haimen City, China. , 2006, Natural toxins.

[2]  C. V. van Dolleweerd,et al.  Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots , 2003, Plant Molecular Biology.

[3]  Carola Engler,et al.  Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors , 2006, Proceedings of the National Academy of Sciences.

[4]  M. Paul,et al.  Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco , 2009, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[5]  J. Fry,et al.  A simple and general method for transferring genes into plants. , 1985, Science.

[6]  G. Borbély,et al.  Microcystin-LR alters the growth, anthocyanin content and single-stranded DNase enzyme activities in Sinapis alba L seedlings. , 2003, Aquatic toxicology.

[7]  T. Lehner,et al.  Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants , 1994, European journal of immunology.

[8]  R. Hellens,et al.  pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation , 2000, Plant Molecular Biology.

[9]  D. Hanahan Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[10]  Y. Ueno,et al.  Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay. , 1996, Carcinogenesis.

[11]  E. Best,et al.  Genetic analysis of Agrobacterium. , 1991, Methods in enzymology.

[12]  W. Preiser,et al.  Fatal microcystin intoxication in haemodialysis unit in Caruaru, Brazil , 1998, The Lancet.

[13]  M. Tarczyńska,et al.  Toxicity of microcystin from cyanobacteria growing in a source of drinking water. , 2004, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[14]  D. Chargelegue,et al.  Transgenic plants expressing antibodies: a model for phytoremediation , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[15]  Linda A. Lawton,et al.  Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water , 2002, Applied and Environmental Microbiology.

[16]  I. Raskin,et al.  Production of recombinant proteins in plant root exudates , 1999, Nature Biotechnology.

[17]  H. Klee,et al.  Improved vectors for plant transformation: expression cassette vectors and new selectable markers , 1987 .

[18]  T. Lehner,et al.  Generation and assembly of secretory antibodies in plants , 1995, Science.

[19]  L. Lawton,et al.  Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library. , 2000, FEMS microbiology letters.

[20]  R. Lerner,et al.  Completing the circle. , 2002, Nature.

[21]  Philip R. Cohen,et al.  Cyanobacterial microcystin‐LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants , 1990, FEBS letters.

[22]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[23]  C. Marusic,et al.  Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies. , 2009, Plant biotechnology journal.

[24]  S. Ho,et al.  Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. , 1989, Gene.

[25]  G. Whitelam,et al.  Synthesis of a Functional Anti–Phytochrome Single–Chain Fv Protein in Transgenic Tobacco , 1992, Bio/Technology.

[26]  L. Lawton,et al.  Detection of the cyanobacterial hepatotoxins microcystins. , 2005, Toxicology and applied pharmacology.

[27]  J. Meriluoto,et al.  Microcystin uptake inhibits growth and protein phosphatase activity in mustard (Sinapis alba L.) seedlings. , 1998, Toxicon : official journal of the International Society on Toxinology.

[28]  E. Keinan,et al.  Herbicide-resistance conferred by expression of a catalytic antibody in Arabidopsis thaliana , 2006, Nature Biotechnology.

[29]  C. Leifert,et al.  Investigations into the inhibitory effects of microcystins on plant growth, and the toxicity of plant tissues following exposure. , 2001, Toxicon : official journal of the International Society on Toxinology.

[30]  M. Montagu,et al.  Assembly of an antibody and its derived antibody fragment inNicotiana andArabidopsis , 1993, Transgenic Research.