Agriculture Reference
In-Depth Information
need.to.develop.more.“universal”.or.versatile.puriication.methods..Technologies.
employing. on. afinity. tags. provide. possibilities. for. such. more. universal. solu-
tion.
169-173
. In. this. strategy,. instead. of. attempting. to. identify. a. speciic. ligand. to.
the.target.protein,.a.highly.selective.afinity.tag.with.known.ligand.is.fused.to.the.
target.protein.by.genetic.engineering..Successful.examples.of.this.strategy.include.
pharmaceutical. proteins. tagged. with. biotin,. histidine,. glutathione.
S
-transferase.
(GST),. and. c-Myc.
174-179
. In. addition,. tags. developed. for. mAb. puriication. (see.
antibody. puriication. discussed. above). such. as. bacteriophage. capsid. proteins,.
S-layer. proteins,. cellulose-binding. domains,. starch-binding. domains,. oleosins,.
and.tobamovirus.nanoparticle.have.also.been.successfully.used.to.purify.vaccines.
and.other.transgenic.proteins.
156,162,180-183
.The.high.afinity.of.the.Fc.fragment.of.
immunoglobulin.to.Protein.A.provides.an.excellent.tag.for.PMP.puriication..As.
a.result,.many.antigen.proteins.have.been.fused.to.the.heavy.chain.of.antibodies.
and. puriied. by. Protein. A. afinity. chromatography.
184,185
. In. fact,. genetic. fusions.
of.antigens.to.mAbs.against.them.have.created.a.new.class.of.highly.potent.vac-
cines.called.RICs.
186
.The.mAb.component.in.RICs.allows.not.only.facile.purii-
cation. but. also. enable. them. to. induce. signiicantly. stronger. immune. responses.
in. host. animals. without. the. help. of. adjuvants.. In. addition. to. providing. a. conve-
nient.puriication.tool,.afinity.tag.often.adds.several.other.beneits.to.the.target.
protein. such. as. enhancing. its. yield,. solubility,. stability,. and. promoting. its. cor-
rect.folding.
184,187
.However,.no.individual.afinity.tag.alone.is.ideal.in.delivering.
all.and.every.possible.beneit..Consequently,.the.tandem.afinity.tag.puriication.
(TAP).or.combinatorial.tagging.strategy.was.developed.to.deliver.the.maximum.
possible.beneit.
175,188-193
.For.example,.a.dual.afinity.His6-MBP.afinity.tag.vec-
tor. was. developed,. in. which. the. MBP. moiety. improves. the. yield. and. enhances.
the. solubility. of. the. recombinant. protein. while. the. His. tag. facilitates. its. purii-
cation.
194,195
. Since. all. tags. have. the. potential. to. interfere. with. the. structure. and.
biological.activity.of.the.target.recombinant.protein,.for.certain.applications,.they.
have.to.be.removed.from.the.puriied.protein.by.site-speciic.protease..Challenges.
still. remain. in. identifying. proteases. with. both. high. eficiency. and. precision. for.
tag. removal.
196,197
. Some. endoproteases. such. as. thrombin,. TEV. protease,. and. 3C.
protease.often. leave.one.or.two. extra.amino.acid.residues.at.their.cleavage. sites.
and.produce.unnatural.N-terminus.on.the.target.protein.
198
.In.contrast,.proteases.
like.enterokinase.and.factor.Xa.have.better.precision.and.leave.target.proteins.with.
their. native. N-termini.
197
. However,. these. enzymes. have. relatively. low. eficiency.
and. routinely. require. high. enzyme. concentrations. and. long. incubation. time,
197
.
which. often. lead. to. nonspeciic. cleavage. at. cryptic. sites.. Regardless,. the. inclu-
sion.of.proteases.in.the.manufacturing.process.should.be.avoided.whenever.it.is.
possible.as.it.would.require.extra.step(s).to.eliminate.the.protease.from.the.inal.
product..This.additional.step(s).would,.in.turn,.increase.the.overall.processing.cost.
and.raise.additional.regulatory.concerns.
196,197
.Consequently,.nonenzymatic.afin-
ity.tag.removal.techniques.have.been.explored.to.avoid.such.problems.
200-202
.For.
example,.intein,.a.self-splicing.protein.element,.has.been.used.as.a.fusion.partner.
of.afinity.tags.to.replace.protease.cleavage.sites.
202
.The.preliminary.results.of.this.
strategy. are. promising.. However,. further. testing. in. high-throughput. settings. is.
necessary.before.it.can.be.applied.for.large-scale.PMP.manufacturing.
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