(448b) Recovery and Purification of Recombinant Butyrylcholinesterase from Nicotiana Benthamiana Plants | AIChE

(448b) Recovery and Purification of Recombinant Butyrylcholinesterase from Nicotiana Benthamiana Plants


Alkanaimsh, S. - Presenter, University of California, Davis
Karuppanan, K. - Presenter, University of California, Davis
McDonald, K. A. - Presenter, University of California, Davis
Nandi, S. - Presenter, University of California
Rodriguez, R. - Presenter, University of California, Davis
and Purification of Recombinant
Butyrylcholinesterase from Nicotiana benthamiana Plants

Salem Alkanaimsh,
Kalimuthu Karuppanan and Karen A. McDonald

Chemical Engineering
and Materials Science, University of California, Davis, CA, 95616

Human butyrylcholinesterase (hBuChE EC is
a 574 amino acid cholinesterase-hydrolyzing enzyme. Organophosphates (OP) are
highly toxic inhibitors of the acetylcholine-hydrolyzing enzymes like acetylcholinesterase.
The resulting accumulation of acetylcholine can lead to respiratory collapse
and death. Current therapies are based on elevating the serum levels of OP
bioscavengers like BuChE. The major limitation of this therapy is high cost,
with plasma-derived hBuChE costing more than $10,000/treatment. Limitations
like cost and availability necessitate an alternative expression platform
capable of large scale, low-cost production of a fully active and efficacious
recombinant hBuChE (rhBuChE). The development of an
effective rhBuChE is a pressing national security
concern in terms of protecting the nation's warfighters and civilian population
from the threat of attack with OP agents. Therapeutic proteins (e.g. hBuChE)
can be produced in plant leaves such as Nicotiana
using transient agroinfiltration. Nicotiana benthamiana represents a
valuable biopharmaceutical protein expression system due to its scalability, ability to glycosylate proteins and elimination
of risk of viral infection. We have a developed a viral expression system based
on Tobacco mosaic Virus (TMV) that
express a FLAG tagged rhBuChE at a level of 10 µg/g fresh weight. Downstream processing is
a major challenge of Nicotiana benthamiana based where it represents 80% of the overall production costs ADDIN EN.CITE
<EndNote><Cite><Author>Wilken</Author><Year>2012</Year><RecNum>170</RecNum><DisplayText><style R.</author><author>Nikolov, Zivko
and purification of plant-made recombinant
plants</keyword><keyword>Protein extraction</keyword><keyword>Bioseparations</keyword><keyword>Process
. Thus, development
of an effective downstream process for purifying rhBuChE
is important. Use of an anti-FLAG affinity resin to capture rhBuChE
gives only a 20% yield recovery. Procainamide resin which tightly binds to cholinesterases can be used to purify the protein and to
enhance the yield percentage. However prior to this chromatography step,
different pretreatment strategies were employed such as pH reduction, ammonium
sulfate fractionation, polyethylene glycol (PEG) fractionation or two phase
aqueous separation of plant extracts to remove plant
pigments and native plant protein (e.g. Rubisco).

 ADDIN EN.REFLIST 1.            Wilken, L.R. & Nikolov, Z.L.
Recovery and purification of plant-made recombinant proteins. Biotechnology Advances 30, 419-433 (2012).