Hairy-root organ cultures for the production of human acetylcholinesterase.
BACKGROUND
Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified.


RESULTS
As a promising alternative, we report the creation of "hairy root" organ cultures derived via Agrobacterium rhizogenes-mediated transformation from human acetylcholinesterase-expressing transgenic Nicotiana benthamiana plants. Acetylcholinesterase-expressing hairy root cultures had a slower growth rate, reached to the stationary phase faster and grew to lower maximal densities as compared to wild type control cultures. Acetylcholinesterase accumulated to levels of up to 3.3% of total soluble protein, ~3 fold higher than the expression level observed in the parental plant. The enzyme was purified to electrophoretic homogeneity. Enzymatic properties were nearly identical to those of the transgenic plant-derived enzyme as well as to those of mammalian cell culture derived enzyme. Pharmacokinetic properties of the hairy-root culture derived enzyme demonstrated a biphasic clearing profile. We demonstrate that master banking of plant material is possible by storage at 4 degrees C for up to 5 months.


CONCLUSIONS
Our results support the feasibility of using plant organ cultures as a successful alternative to traditional transgenic plant and mammalian cell culture technologies.
Woods
Ryan R
RR
School of Life Sciences and The Biodesign Institute, PO Box 874501, Arizona State University, Tempe, AZ 85287-4501, USA. Ryan.R.Woods@asu.edu
Geyer
Brian C
BC
Mor
Tsafrir S
TS
D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000302:isolation & purification; D000818:Animals; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D051379:Mice; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D000110:Acetylcholinesterase