Application of protein engineering

Protein Engineering

1. APPLICATION OF PROTEIN ENGINEERING
AKSHAY PARMAR
Post Graduate Student | Department Of Biochemistry
School Of Science,
Gujarat University.

2. WHAT IS PROTEIN ENGINEERING ?
•Proteinengineeringcanbedefinedasthemodificationofprotein
structurewithrecombinantDNAtechnologyorchemicaltreatmentto
getadesirablefunctionforbetteruseinmedicine,industryand
agriculture.
•Proteinengineeringistheprocessofdevelopingusefulor
valuableproteins.Itisayoungdiscipline,withmuchresearchtaking
placeintotheunderstandingofproteinfoldingandrecognition
forproteindesignprinciples.Itisalsoaproductandservicesmarket,
withanestimatedvalueof$168billionby2017.

3. FOODINDUSTRY
•Importantapplicationareaofproteinengineeringregardingfoodindustryis
thewheatglutenproteins.Theirheterologousexpressionandprotein
engineeringhasbeenstudiedusingavarietyofexpressionsystems,suchas
E.coli,yeastsorculturedinsectcells.
•Wildtypeandmutantwheatglutenproteinswereproducedtocomparethem
toeachotherforproteinstructure-functionstudiesbecauseoftheir
availability,rapidandeasyuse,aswellashighexpressionlevels.
•Foodindustrymakesuseofavarietyoffood-processingenzymes,suchas
amylasesandlipases,thepropertiesofwhichareimprovedusing
recombinantDNAtechnologyandproteinengineering.Thedeletionofnative
genesencodingextracellularproteases,forexample,increasedenzyme
productionyieldsofmicrobialhosts.

4. •SomelargegroupsofenzymeslikeProteases,AmylasesandLipasesare
importantforbothfoodanddetergentindustries,astheyhaveabroadrangeof
industrialapplications.
•Proteasesareusedinseveralapplicationsoffoodindustryregardinglow
allergenicinfantformulas,milkclottingandflavors.Theyarealsoimportant
fordetergentindustryforremovingproteinstains.
•The improvement of proteases for industry to have, for example, high activity
at alkaline pH and low temperatures, or improved stability at high
temperatures is a challenge for protein engineering.
•Microbial protease production is industrially suitable because of low costs,
high production yields , and easy genetic manipulation.
•Amylasesare also important for both food and detergent industries. In food
industry, they are use for liquefaction and saccharificationof starch, as well as
in adjustment of flour and bread softness and volume in baking.

5. •Thedetergentindustrymakesuseofamylasesinremovalofstarchstains
Recently,theproductionof“functionalfoods”isbecomingincreasingly
importantforfoodindustry.Particularly,theproductionofindustrialproducts
andfunctionalfoodsfromcheapandrenewablerawagriculturalmaterialsis
desirable.
•Conversionofstarchtobioethanolortofunctionalingredientslikefructose,
wine,glucoseandtrehalose,requiresmicrobialfermentationinthepresence
ofbiocatalystssuchasamylasestoliquefyandsaccharifystarch.Toimprove
theindustriallyimportantpropertiesofamylases,suchashighactivity,high
thermo-andpH-stability,highproductivity,etc.;recombinantenzyme
technology,proteinengineeringandenzymeimmobilizationhavebeenused.
•Anothermajorgroupofenzymesutilizedbyfoodanddetergentindustriesis
constitutedbyLipases.

6. •Lipasesareusedinmanyapplicationsoffoodindustrysuchasforthe
stabilityandconditioningofdough(asaninsituemulsifier),andincheese
flavourapplications.
•Lipasesarealsocrucialforthedetergentindustry,astheyareusedin
removaloflipidstainsAslipasesarecommonlyusedinfoodindustrial
applications,havingtoxicologicallysafelipasesisanimportantrequirement
offoodindustry.

7. ENVIROMENTAL APPLICATIONS
•Environmentalapplicationsofenzymeandproteinengineeringarealso
anotherimportantfield.
•Geneticmethodsandstrategiesfordesigningmicroorganismstoeliminate
environmentalpollutantsandincludedgeneexpressionregulationto
providehighcatalyticactivityunderenvironmentalstressconditions,such
asthepresenceofatoxiccompound,rationalchangesintroducedin
regulatoryproteinsthatcontrolcatabolicactivities,creationofnew
metabolicroutesandcombinations.
•Manyorganicpollutantssuchasphenols,azodyes,organophosphorus
pesticidesandpolycyclicaromatichydrocarbonscanbedetoxifiedusing
enzymaticoxidation.

8. •Petroleumbiorefiningisalsoanimportantenvironmentalapplicationarea,
wherenewbiocatalystsarerequired.
•Proteinengineering,isolationandstudyofnewextremophilic
microorganisms,geneticengineeringdevelopmentsareallpromising
advancestodevelopnewbiocatalystsforpetroleumrefining.Petroleum
biorefiningapplicationssuchasfuelbiodesulfurization,denitrogenationof
fuels,heavymetalremoval,depolymerisationofAsphaltenes.
•Manyproteinengineeringstrategieswereidentifiedsuchasimprovement
ofhydrogenperoxidestability,increasingtheredoxpotentialtobroaden
thesubstraterange,heterologousexpressionandindustrialproduction
development.

9. MEDICAL APPLICATION
•Theuseofproteinengineeringforcancertreatmentstudiesisamajorareaof
interest.Pretargetedradioimmunotherapyhasbeendiscussedasapotential
cancertreatment.Bypretargeting,radiationtoxicityisminimizedby
separatingtherapidlyclearedradionuclideandthelong-circulatingantibody.
•AdvancesinproteinengineeringandrecombinantDNAtechnologywere
expectedtoincreasetheuseofpretargetedradioimmunotherapy.
•Theuseofnovelantibodiesasanticanceragentsisalsoanimportantfieldof
application,wheretheabilityofantibodiestoselectantigensspecificallyand
withhighaffinityisexploited,andproteinengineeringmethodsareusedto
modifyantibodiestotargetcancercellsforclinicalapplications.
•Recently,theterm“modularproteinengineering”hasbeenintroducedfor
emergingcancertherapies.

10. •Treatmentstrategiesbasedontargetednanoconjugatestobespecifically
directedagainsttargetcellsarebecomingincreasinglyimportant.
Additionally,multifunctionalandsmartdrugvehiclescanbeproducedat
thenanoscale,byproteinengineering.Thesestrategiescouldbecombined
toidentifyandselecttargetsforprotein-baseddrugdelivery.
•Pharmacokineticpropertiesofantibodieshavebeenimprovedbyprotein
engineeringandantibodyvariantsofdifferentsizeandantigenbindingsites
havebeenproducedfortheultimateuseasimagingprobesspecifictotarget
tissues.
•Molecularimagingtoolsbasedonantibodieswillfindmoreapplicationsin
thefutureregardingdiagnosisandtreatmentofcancerandothercomplex
diseases.

11. BIOPOLYMER PRODUCTION
•Proteinengineeringapplicationsforbiopolymerproductionarealso
promising.Particularly,peptidesarebecomingincreasinglyimportantas
biomaterialsbecauseoftheirspecificphysical,chemicalandbiological
properties.
•Proteinengineeringandmacromolecularselfassemblyareutilizedto
producepeptide-basedbiomaterials,suchaselastin-likepolypeptides,silk-
likepolymers.
•Theabilityofproteinengineeringtocreateandimproveproteindomainscan
beutilizedforproducingnewbiomaterialsformedicalandengineering
applications.

12. NANOBIOTECHNOLOGY
•Nanobiotechnologyapplicationsofproteinengineeringarebecoming
increasinglyimportant.Thesynthesisandassemblyofnanotechnological
systemsintofunctionalstructuresanddeviceshasbeendifficultandlimiting
theirpotentialapplicationsforalongtime.
•Biologicalmacromolecules,suchasproteins,carbohydratesandlipidsare
usedinthesynthesisofbiologicaltissuesinaqueousenvironmentsandmild
physiologicalconditions,wherethisbiosyntheticprocessisundergenetic
regulation.
•Particularlyproteinsarecrucialelementsofbiologicalsystems,basedon
theirrolesintransport,regulationoftissueformation,physicalperformance
andbiologicalfunctions.Thus,theyaresuitablecomponentsforcontrolled
synthesisandassemblyofnanotechnologicalsystems.

13. •Combinatorialbiologymethodscommonlyappliedinproteinengineering
studies,suchasphagedisplayandbacterialcellsurfacedisplay
technologies,arealsousedtoselectpolypeptidesequenceswhich
selectivelybindtoinorganiccompoundsurfaces,forultimateapplications
ofnanobiotechnology.
•Anotherinterestingnanotechnologyapplicationistheuseofamyloidfibrils
asstructuraltemplatesfornanowireconstruction.Thisapplicationisbased
onthefactthatsomeproteinsformwell-orderedfibrillaraggregatesthat
arecalledamyloidfibrils.
•Theselfassociationofwell-orderedgrowthfibrilsthroughnoncovalent
bondsundercontrolledconditionswassuggestedtohaveahighpotentialto
beusedfornanobiotechnology.Theuseofamyloidfibrilsasstructural
templatesfornanowireconstruction.

14. REDOX PROTEINS AND ENZYMES
•Suchproteinsandenzymescanbemodifiedtobeusedinnanodevicesfor
biosensing,aswellasfornanobiotechnologyapplications.
•Theelectrochemistryofredoxproteinsparticularlydrawsattentionfor
applicationsinbiofuelcells,chemicalsynthesisandbiosensors.
•Proteinengineeringofredox-activeenzymespointedouttwoemergingareas
ofproteinengineeringofredox-activeenzymes:novelnucleicacid-based
catalystconstruction,andintra-molecularelectrontransfernetwork
remodelling.
•CytochromeP450monooxygenasesandproteinengineeringofcytochrome
P450biocatalystsformedical,biotechnologicalandbioremediation
applications.

15. OTHER NEW APPLICATION
•“Insertionalproteinengineering”applicationsarealsobecomingimportant,
particularlyforbiosensorstudies.Theapplicationsofinsertionalprotein
engineeringforanalyticalmolecularsensing.
•“Zincfingerproteinengineering”isanotherapproachthathasbeenusedin
generegulationapplications.Thezincfingerdesignandprincipleisusedto
designDNAbindingproteinstocontrolgeneexpression.
•“Virusengineering”isanotheremergingfield,wherethevirusparticlesare
modifiedbyproteinengineering.Viruseshavemanypromisingapplications
inmedicine,biotechnologyandnanotechnology.Theycouldbeusedasnew
vaccines,genetherapyandtargeteddrugdeliveryvectors,molecularimaging
agentsandasbuildingblocksforelectronicnanodevicesornanomaterials
construction.

16. •“Proteincysteinemodifications”arealsoimportantproteinengineering
applications.Ascysteinemodificationsinproteinscausediversitiesin
proteinfunctions,cysteinethiolchemistryhasbeenappliedforinvitro
glycoproteinsynthesis.
•Thismethodcouldbepotentiallyusedfordevelopmentofnewprotein-
baseddrugs,improvingtheirhalf-life,reducingtheirtoxicityand
preventingmultidrugresistancedevelopment.

17. In Many Biological Structures Proteins Are
Simply Components Of Larger Molecular
Machines.
-Michael Behe(American Biochemistry)

18. Thank YOU