Expression and Manufacture of Pharmaceutical Proteins in Genetically Engineered Horticultural Plants - Transgenic Horticultural Crops: Challenges and Opportunities - page 86

Pharmaceutical protein gene

Integrate into a
viral coding sequence
for expression as a
“by product” of
viral replication

Incorporate into
deconstructed viral
sequences as part
of Agrobacterium
transformation
vector

Clone into plant
transformation
vector

Transient expression:
high-level protein
expression without
forming infectious viral
particles

Transient expression:
infect plant to initiate
viral replication

Stable expression:
regulated release of
viral replicon from a
viral genome integrated
into plant genome

Stable expression:
integrate into nuclear
genome, high-level
protein expression

Stable expression:
transgene
integrates into
chloroplast genome

Stable expression:
transgene
integrates into
nuclear genome

FIGURE 4.1 Strategies. for. the. expression. of. pharmaceutical. proteins.in. plants..(Adapted.
from.Chen,.Q.,. Biol. Eng .,.1,.291,.2008..With.permission.)

the. stable. transformation. of. the. chloroplast. genome,. and. transient. transformation.
(Figure.4.1)..In.the.irst.two.strategies,.an.expression.cassette.harboring.the.gene.of.
interest.is.used.to.transform.plant.cells.to.enable.the.insertion.of.transgene.into.
the.nuclear.or.chloroplast.genomes..The.stable.integration.of.the.cassette.into.either.the.
nuclear. or. plastid. genome. allows. the. Mendelian. inheritance. of. the. transgene. over.
generations.and,.in.turn,.the.stable.expression.of.transgenic.proteins..The.resulting.
transgenic.lines.can.be.propagated.to.establish.a.master.seed.bank.for.future.large-
scale. production. of. pharmaceutical. proteins.. For. most. plant. species,. exogenous.
transgene.cassette.can.be.delivered.into.their.genome.by.using. Agrobacterium tume-
faciens ,.which.inserts.DNA.into.the.plant.genome.in.a.somewhat.random.fashion..
Alternatively,. ballistic. methods. with. DNA-coated. microprojectiles. can. be. used. to.
bombard.plant.cells..The.ballistic.“gene.gun”.is.especially.useful.for.plant.species.
that.are.dificult.to.transform.eficiently.by. Agrobacterium . 15 .Up.to.today,.the.bal-
listic.method.with.tungsten.or.gold.particles.remains.to.be.the.only.eficient.way.for.
the.transformation.of.the.chloroplast.genome.
In. the. third. plant. expression. strategy,. transgenes. are. not. integrated. into. one. of.
the.plant.genomes,.but.instead.they.are.present.in.the.plant.nucleus.transiently.while.
being.transcribed,. and. later.the.transcripts.are.transported.into.the.cytoplasm.and.
the.transgenic.proteins.are.translated..While.transient.expression.can.be.carried.out.
with. both. nonviral. and. viral. vectors,. the. latter. are. more. widely. used. due. to. their.
robustness. in. replication,. transcription,. and. translation. 16 . Plant. viral. vectors. have.
been.used.in.several.major.formats.to.transiently.express.PMPs..For.example,.many.
vaccine.epitopes.have.been.fused.to.the.viral.coat.protein.so.that.they.will.be.dis-
played. of. on. the. surface. of. the. virus. 17,18 . PMP. genes. can. also. be. inserted. into. an.