Biomedical Engineering Reference
In-Depth Information
chromophores. (naphthalene,. stilbene,. and. oligo-
p
-phenylenevinylene). (de. la. Escosura.
et. al.. 2010).. Oligothymine. mixed. with. coat. protein. produce. spherical. T.=.1. particles;.
however,.upon.adding.the.chromophores.(which.base-pair.with.thymine.and.change.the.
stiffness),.rod-like.structures.formed.that.could.be.over.1.μm.long..It.is.clear.from.these.
observations. that. CCMV. coat. proteins. have. template-dependent. assembly. behavior,.
which.open.up.new.possibilities.for.materials.engineering.
The. self-assembly. properties. of. the. CCMV. coat. protein. can. also. be. used. to. encapsu-
late.hydrophobic.compounds..In.one.approach,.oil.droplets.stabilized.by.sodium.dodecyl.
sulfate.(SDS).have.been.“decorated”.by.CCMV.coat.protein.(Chang.et.al..2008)..Although.
SDS. particles. were. larger. than. the. native. CCMV. capsid,. the. hexameric. capsomer. units.
are.observed.assembling.over.the.surface..In.a.similar.approach,.Kwak.et.al..(2010).used.
DNA-based.amphiphiles.to.produce.VLPs.with.a.hydrophobic.core.to.load.hydrophobic.
compounds..Short.11-.or.22-mer.DNA.sequences.with.a.hydrophobic.tail.were.loaded.with.
pyrene,.which.upon.mixing.with.CCMV.coat.protein.at.pH.7.5.formed.∼20.nm.particles.
(T.=.2).. Multifunctional. VLPs. were. demonstrated. in. this. system. by. hybridizing. a. luo-
rescent.ROX-DNA.conjugate.to.the.DNA-micelle,.prior.to.adding.the.coat.protein..About.
25.micelles.were.packaged.into.the.construct.with.this.approach.
Perhaps. one. of. the. most. intriguing. developments. using. CCMV. assembly. is. the. pos-
sibility.to.incorporate.other.proteins.inside.the.VLP.core..Comellas-Aragonés.et.al..(2007).
demonstrated.for.the.irst.time.in.2007.that.a.single.horseradish.peroxidase.(HRP).enzyme.
can.be.internalized.and.remain.active..Their.approach.was.to.simply.mix.HRP.with.coat.
protein. and. use. the. conventional. pH. assembly. process. to. entrap. the. enzyme. (initially.
mixing.at.pH.7.5.and.then.dialyzing.against.lower.pH.buffer)..Single-enzyme.encapsula-
tion. was. achieved. by. manipulating. the. coat. protein:HRP. ratio.. It. is. useful. to. be. able. to.
manipulate.the.amount.of.protein.being.encapsulated;.however,.it.could.be.hard.to.control.
this. relying. solely. on. increasing. concentration.. One. way. to. improve. the. encapsulation.
eficiency. is. to. incorporate. self-recognizing. peptides. in. the. system.. Pair. motifs. of. self-
assembling.short.coiled-coil.peptides.(seven.amino.acids).are.frequently.found.in.nature.
and. tend. to. associate. with. high. afinity.. Minten. et. al.. (2009a,2010). used. a. heterodimeric.
coiled-coil. pair. to. realize. controlled. encapsulation. of. green. luorescent. protein. (GFP). in.
CCMV. VLPs.. They. fused. a. short. positively. charged. peptide. (K-coil). to. the. coat. protein.
while. GFP. were. modiied. to. contain. a. complimentary. negatively. charged. coil. (E-coil).
(Figure.7.4)..Upon.mixing.the.recombinant.proteins.at.pH.7.5.and.then.lowering.pH.to.5.0,.
pH 7.5
pH 7.5
pH 5.0
+
Capsid dimer
with K-coil
EGFP
with E-coil
EGFP-capsid protein
complex
Wild-type
capsid protein Capsid with EGFP
Figure 7.4
(See companion CD for color igure.)
. CCMV.coat.protein.and.GFP.with.K/E-peptides,.respectively..Coiled-
coil.formation. followed. by. VLP.assembly. encapsulates. GFP. with. consistent. results.. (Reprinted. with. permis-
sion.from.Minten,.I.J.,.Hendriks,.L.J.A.,.Nolte,.R.J.M.,.Cornelissen,.J.J.L.M.,.Controlled.encapsulation.of.multiple.
proteins.in.virus.capsids,.
J. Am. Chem. Soc
.,.131(49),.17771-17773..Copyright.2009a.American.Chemical.Society.)
