Biomedical Engineering Reference
In-Depth Information
the. lactam. antibiotic. cefotaxime.. The. designed. scaffold. modiications. in. this. study. were.
inspired.by.MBL,.which.belongs.to.the.same.structural.superfamily.as.GlyII.and.provided.
an. example. solution. for. the. design. of. a. β-lactamase.. While. the. successful. conversion. of.
GlyII.into.a.β-lactamase.is.remarkable,.the.application.of.this.approach.to.the.generation.of.
novel.artiicial.enzymes.is.limited.by.the.requirement.for.a.homologous.natural.enzyme.
3.3 Directed Evolution with Rational Library Design
The. absence. of. any. requirement. for. structural. or. functional. knowledge. is. a. strength.
of. directed. evolution.. In. addition. to. circumventing. our. ignorance. of. protein. structure/
function.relationships,.random.mutagenesis.and.screening.often.inds.beneicial.mutations.
at.positions.far.from.the.active.site.that.could.not.have.been.predicted.rationally..However,.
random.approaches.rely.on.the.ability.to.screen.large.numbers.of.mutants,.and.inding.a.
needle.in.the.haystack.requires.considerable.luck..In.addition,.while.activities.present.at.
low.levels.in.the.starting.protein.can.be.improved,.generating.entirely.new.activity.is.very.
dificult..Researchers.are.developing.a.variety.of.strategies.to.address.these.limitations.by.
using.rational.methods.to.enrich.mutant.libraries.in.active.mutants.
A. simple. but. extremely. successful. rational. library. design. method. has. been. the.
application. of. structural. data. to. the. selection. of. crossover. points. during. recombination-
based.library.generation..“Sexual”.recombination.of.homologous.genes.takes.advantage.
of.sequence.diversity.already.vetted.by.nature.for.compatibility.with.a.particular.fold.and.
function.. However,. random. recombination. can. introduce. clashes. and. disrupt. important.
contacts,. lowering. the. fraction. of. library. members. that. fold. successfully.. The. SCHEMA.
(Voigt.et.al..2002).and.Recombination.as.a.Shortest.Path.Problem.(RASPP;.Endelman.et.al..
2004).protocols.analyze.structural.contacts.to.determine.positions.at.which.recombination.
is. least. likely. to. disrupt. the. structure.. The. resulting. designed. libraries. are. enriched. in.
folded.proteins.and.have.been.shown.to.outperform.libraries.generated.by.random.DNA.
shufling..These.methods.have.been.used.to.generate.high-quality.libraries.of.P450.heme.
proteins.(Otey.et.al..2006).and.fungal.cellulases.(Heinzelman.et.al..2009).
Multiple.sequence.alignments.of.homologous.proteins.can.be.used.in.library.generation.
to.provide.information.about.which.amino.acids.are.allowed.at.each.position,.narrowing.
down. the. size. of. the. sequence. space. to. be. searched.. Bias. from. the. evolutionary. history.
of.these.sequences.can.be.removed.statistically.(Halabi.et.al..2009).or.avoided.entirely.by.
selecting.competent.sequences.from.synthetic.pools.(Jäckel.et.al..2010)..Compatible.diver-
sity.at.each.site.is.then.built.into.synthetic.degenerate.oligonucleotides,.which.are.assem-
bled.by.PCR.to.yield.a.diverse.collection.of.mutant.genes..Designed.libraries.can.also.be.
constructed. so. as. to. preserve. the. correlations. between. amino. acid. identities. at. multiple.
positions..One.such.library.(Lippow.et.al..2010),.which.maintained.the.linkages.between.
neighboring.amino.acid.identities.in.a.computationally.redesigned.active.site,.was.shown.to.
be.enriched.in.active.mutants.relative.to.a.control.library.with.no.interposition.information.
In. an. example. of. how. artiicial. enzymes. could. revolutionize. the. chemical. industry,.
scientists. at. Codexis. created. an. enzyme. to. replace. a. high-pressure,. rhodium-catalyzed.
asymmetric.hydrogenation.step.in.the.synthesis.of.sitagliptin,.an.antidiabetic.pharmaceu-
tical.(Savile.et.al..2010)..A.homology.model.(model.of.a.protein.with.unknown.3D.struc-
ture.based.on.sequence.similarity.to.a.known.protein).of.a.transaminase.enzyme.with.no.
activity.for.the.prositagliptin.ketone.indicated.positions.in.the.binding.pocket.that.could.
