Biomedical Engineering Reference
In-Depth Information
VLPs.containing.as.many.as.∼15.GFPs.could.be.obtained.consistently..In.addition.to.GFP,.
lipase. B. from.
Pseudozyma antarctica
. has. also. been. packaged. with. this. approach. to. get. a.
catalytically.active.VLP.(Minten.et.al..2011a)..The.pH-dependent.assembly.of.CCMV.is.one.
reason.why.it.is.attractive.as.a.nanocontainer;.however,.above.pH.∼7.5,.it.is.not.functional.
as.it.will.disassemble.into.dimers..In.an.attempt.to.remedy.this,.Minten.et.al.,.as.a.continu-
ation.of.their.work,.have.included.a.coat.protein.with.an.N-terminal.His-tag.in.addition.to.
the.coiled-coil.peptides..After.VLPs.are.formed.at.pH.5,.Ni
2+
.is.added.to.the.solution,.which.
causes.the.His-tags.to.associate.and.stabilize.the.structure.when.increasing.the.pH.to.7.5.
(Minten.et.al..2011b)..Improving.the.pH.stability.of.CCMV.VLPs.opens.up.many.possibili-
ties.in.various.application.areas.
7.3 M13 Bacteriophage
7.3.1 general Properties, Structure, and Assembly
The.M13.bacteriophage.has.been.of.great.importance.to.both.ields.of.molecular.biology.
and.nanoengineering..The.use.of.its.circular.single-stranded.DNA.genome.in.cloning.
applications.aided.the.advancement.of.DNA.sequencing.through.the.use.of.
Escherichia
coli
. as. an. ampliication. vector. (Messing. et. al.. 1993).. Later,. the. development. of. phage.
display,. a. technique. in. which. foreign. peptides. are. expressed. on. the. coat. protein. of.
M13,.enabled.a.revolution.in.screening.peptide.sequences.for.high-afinity.target.bind-
ing,. recombinant. antibody. technology,. and. more. recently,. nanostructure. assembly.
(Rakonjac. et. al.. 2011).. While. the. concept. of. phage. display. has. had. an. immeasurable.
impact. in. biotechnology,. it. is. only. given. cursory. treatment. here,. as. it. is. beyond. the.
scope. of. this. text.. We. refer. the. reader. to. other. references. that. treat. phage. display. in.
more. detail. for. additional. information. (Georgieva. et. al.. 2011,. Pande. et. al.. 2010,. Smith.
and.Petrenko.1997).
The. M13. bacteriophage. is. a. ilamentous,. nonenveloped. virus. in. the. family.
Inoviridae
..
It.infects.bacteria,.beginning.with.binding.to.the.F.pili.of.
E. coli
,.and.is.a.Biosafety.Level.
1. pathogen.. Interestingly,. M13. infection. of. bacteria. is. not. virulent,. that. is,. secretion. of.
progeny.phage.from.the.infected.host.does.not.cause.lysis,.allowing.continued.host.cell.
division.and.M13.production..The.M13.capsid.exhibits.ivefold.helical.symmetry.and.is.
approximately.7.nm.wide.and.900.nm.long..The.mature.capsid.consists.of.ive.different.
proteins,.and.a.single,.circular.strand.of.single-stranded.DNA,.6407.nucleotides.in.length..
Figure.7.5.shows.the.structure.of.the.M13.bacteriophage..The.DNA.strand,.although.dou-
bled-back.on.itself.due.to.its.circular.nature,.does.not.exhibit.Watson-Crick.base.pairing..
The.result.is.a.nonspeciic.interaction.between.the.genome.and.the.coat.protein,.leading.
to.a.varying.ratio.between.the.number.of.nucleotides.and.protein.coat.monomers.in.the.
virion..In.fact,.the.length.of.the.DNA.segment.contained.within.the.capsid.can.determine.
virion.length,.with.capsids.as.short.as.50.nm..The.length.of.the.mature.virion.is.made.up.
of. approximately. 2700. copies. of. the. major. coat. protein,. pVIII,. a. 50-amino. acid. peptide.
that. is. the. most. commonly. used. target. in. phage. display.. The. ends. of. the. ilamentous.
capsid.are.made.up.of.ive.pairs.of.the.remaining.major.proteins,.pIII-pVI,.and.pVII-pIX.
(Rakonjac.et.al..2011).
Self-assembly. of. M13. occurs. in. the. cytoplasm. at. the. inner. membrane. of. the. host. cell..
Phages.are.not.produced.and.then.subsequently.released;.rather.they.are.extruded.from.
the.host.cell.membrane.during.assembly..The.packaging.signal.in.the.DNA.sequence.is.a.
