Biomedical Engineering Reference
In-Depth Information
be.targeted.for.mutagenesis..Site.saturation.mutagenesis.and.screening.identiied.a.vari-
ant.with.four.mutations.that.had.low.activity.toward.the.substrate..Additional.rounds.of.
directed.evolution.under.industrially.realistic.conditions.resulted.in.an.activity.improved.
by.four.orders.of.magnitude.and.an.enantiomeric.excess.of.>99.95%,.along.with.the.abil-
ity. to. function. in. the. very. unnatural. environment. of. a. bioreactor:. 50%. organic. solvents.
(required.to.keep.the.substrates.in.solution),.40°C,.and.250.mM.substrate..Even.under.these.
harsh.conditions,.the.enzyme.remained.stable.for.more.than.24.h,.demonstrating.that.pro-
tein.as.a.nanotechnological.substrate.is.by.no.means.limited.to.natural.environments.
Statistical.methods.and.machine.learning.are.increasingly.applied.to.isolate.the.effects.
of. individual. mutations. on. stability. and. function. and. predict. optimal. combinations. of.
mutations.. Statistical. approaches. can. be. particularly. valuable. when. no. high-throughput.
screen.is.available,.and.are.becoming.more.attractive.as.the.economics.of.DNA.sequenc-
ing.and.synthesis.improve..By.analogy.to.the.quantitative.structure-activity.relationship.
method. popular. in. drug. development,. an. algorithm. based. on. protein. sequence-activity.
relationships.(ProSARs).has.been.developed.and.applied.to.enzyme.engineering.(Fox.et.al..
2003)..In.ProSAR,.mutants.are.screened.and.sequenced,.and.the.effect.of.each.individual.
mutation.is.resolved.by.partial.least-squares.regression..New.mutations.are.added.to.the.
pool. as. beneicial. mutations. are. identiied. and. deleterious. mutations. are. removed. from.
consideration..ProSAR.was.used.to.eficiently.adapt.halohydrin.dehalogenase.mutants.to.
the. demands. of. an. industrial. process. for. the. production. of. the. starting. material. for. the.
cholesterol.drug,.Lipitor.(Fox.et.al..2007)..A.similar.DNA.synthesis-based.strategy.involves.
synthesizing.small.collections.of.mutants.containing.combinations.of.promising.mutations.
identiied.via.analysis.of.alignments.of.homologous.sequences.and.then.teasing.apart.the.
contributions.of.each.mutation.to.the.performance.of.a.small.number.of.mutants..Testing.
a.total.of.fewer.than.100.custom-synthesized.mutants.over.two.rounds.of.library.construc-
tion.provided.enough.data.to.construct.proteinase.K.variants.with.20-fold.improvements.
in.thermostability.(Liao.et.al..2007)..A.similar.approach.identiied.ive.mutations.to.prolyl.
endopeptidase.that.increased.the.stability.of.the.enzyme.under.gastric.conditions,.again.
requiring.the.synthesis.and.screening.of.fewer.than.100.mutants.(Ehren.et.al..2008).
A.particularly.exciting.new.extension.of.these.ideas.is.the.use.of.next-generation.sequenc-
ing. technologies. to. generate. itness. landscapes. of. the. WW-domain. protein. (Fowler. et. al..
2010),. an. RNA. ligase. ribozyme. (Pitt. and. Ferre-D'Amare. 2010),. and. the. chaperone. Hsp90.
(Hietpas.et.al..2011)..Deep.sequencing.was.used.to.identify.the.sequences.of.those.members.
of.a.mutant.library.deemed.active.by.a.functional.selection..The.abundance.of.each.sequence.
in.the.selected.pool.was.taken.as.a.measure.of.the.itness.of.that.sequence..The.copious.data.
that.result.from.this.method.form.a.detailed.sequence/activity.itness.landscape.that.iden-
tify.sites.critical.for.folding.and.function.and.inform.further.rounds.of.library.design.
3.4 Selection of Enzymes with No Natural Parent
An.interesting.new.approach.to.library.construction.uses.rational.or.computational.design.
over.the.entire.protein.to.restrict.the.library.to.sequences.likely.to.fold.into.stable.structures.
while.preserving.as.much.diversity.as.possible..This.approach.leaves.the.catalytic.mecha-
nism.up.to.chance,.merely.restricting.the.set.of.possible.amino.acids.at.each.site.to.those.
likely.to.be.compatible.with.a.desired.three-dimensional.structure..While.this.approach.
generates.libraries.too.large.to.exhaustively.screen,.active.proteins.can.be.isolated.when.
