PRODUCTION OF PLANT DEFENSE PROTEINS FROM
TRICHOSANTHES KIRILOWII PLANT CELL CULTURES
Karen A. McDonald, N.-J. Remi Shih, and Alan P.
Jackman
University of California, Davis
Department of Chemical Engineering and Materials Science
Davis, CA 95616
We are studying the production of plant defense proteins from
plant cell suspension cultures in shake flasks and bioreactors.
Of particular interest are the ribosome-inactivating proteins
(RIPs) (see reviews by Barbieri et al. (1993), Girbes et al.
(1996), Hartley et al. (1996) and Stirpe et al (1992)), specific
rRNA glycosidases, which remove adenine at a well conserved site
on ribosomal RNA causing the shut down of protein synthesis in
both eucaryotic and procaryotic cells. RIPs have potential
applications as antiviral therapeutics (McGrath et al., 1989),
immunotoxins for cancer therapies, and genetic engineering of
crops for improved disease resistance (Lodgeman et al., 1992).
In our laboratory we are studying the production of RIPs from
plant cell suspension cultures of Trichosanthes kirilowii,
a member of the Cucurbitaceae family found in Japan, Korea and
China. We have purified and characterized 4 novel RIPs, ranging
from 15 kDa to 35 kDa in size, from the plant cell culture
broths; these proteins possess the specific rRNA N-glycosidase
activity which is characteristic of RIPs and inhibit protein
synthesis in cell free translation systems.
We have also investigated the kinetics of growth and RIP production of T. kirilowii plant cell suspension cultures in 5 L agitated, sparged bioreactors under well controlled conditions of temperature (27 C), dissolved oxygen concentration (70% sat), and agitation (50-100 rpm using a pitched blade impeller).
We have found that the cultures reached final biomass
concentrations as high as 20 g dw/L and a doubling time of less
than 2 days during the exponential growth phase. The lag phase
for these cultures was variable, ranging from 4-11 days,
depending on the inoculum source and inoculation density.
Extracellular RIP production was observed in the culture broths during the exponential growth phase and reaches a level of 50-58 units of activity (1 unit = protein concentration causing 50% inhibition of protein synthesis in a cell-free translation system using rabbit reticulocyte lysate), however, RIP activity declined during the stationary phase presumably due to proteolytic degradation. Sample results for extracellular RIP production for one of our batch cultures are shown in the figure below
Work is currently underway to purify and characterize the
extracellular RIPs from these cultures. We are also investigated
alternative bioprocessing strategies for enhanced production of
RIPs from plant cell cultures.
REFERENCES
Barbieri L; Battelli MG; Stirpe F. Ribosome-Inactivating
Proteins From Plants, Biochimica Et Biophysica Acta 1993,
1154: 237-282.
Girbes T.; Ferreras JM.; Iglesias R.; Citores L.; Detorre C.;
Carbajales ML.; Jimenez P.;, Debenito FM.; Munoz R. Recent
Advances In The Uses And Applications Of Ribosome-Inactivating
Proteins From Plants, Cellular And Molecular Biology 1996,
42: 461-471.
Hartley MR; Chaddock JA; Bonness MS. The Structure And
Function Of Ribosome-Inactivating Proteins, Trends In Plant
Science 1996, 1: 254-260.
Logemann, J.; Jach, G.; Tommerup, H.; and Mundy, J. Expression
of a Barley Ribosome-Inactivating Protein Leads to Increased
Fungal Protection in Transgenic Tobacco Plants. Bio/Technology,
1992, 10:305-308.
McGrath, M.S.; Hwang, K.M.; Caldwell, S.E.; Gaston, I.; Luk,
K.C.; Ledas, P.V.; Vennari, J.C.; Yeung, H.W.; Lifson, J.D. An
Inhibitor of Human Immunodeficiency Virus Replication in Acutely
and Chronically Infected Cells of Lymphocyte and Mononuclear
Phagocyte Lineage, Proc. Natl. Sci. USA 1989,
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Stirpe F; Barbieri L; Battelli MG; Soria M; Lappi DA.
Ribosome-Inactivating Proteins From Plants - Present Status And
Future Prospects, Bio/Technology 1992, 10: 405-412.
Presenting author: McDonald, Karen
e-mail: kamcdonald@ucdavis.edu
http: www.engr.ucdavis.edu/~pcdhome/karen/Research.htm
