Protein Engineering

It describes the Rational and Random approaches of protein engineering and various methods of modifications in protein structure.

1. PROTEIN ENGINEERING
Mr. A. T. Sharma
Assist. Professor
Nanded Pharmacy College, Nanded

2. Introduction
•Definition:Processofdevelopingproteinswith
desiredfunctionsbymanipulatingstabilityand
specificityofproteins.
•Methodofdevelopingusefulandvaluable
proteins.
•Methodofchangingaminoacidsequenceina
proteintoachieveadesiredresult.E.g.changein
substratespecificity,increasedstabilityto
temperaturechanges,organicsolvents,extreme
pH

3. Modificationinaminoacidsequencecan
bedoneby-
•Oneormorecodonwillcodefordifferentaminoacidtype
•Insertionsanddeletionsofentirecodons
•Chemicalmodificationsofproteins
➢Resultantproteins–same3Dstructureasofnativeprotein
➢Modificationsinsequenceininteriorofproteins–
deleteriouseffectonproteinfunction
➢Aminoacidsarecloselypacked–stearichindrance–
changein3Dstructure–functionalchanges–impaired
proteinactivity
➢Hencesurfacelocationsmodifications

4. Approaches
•Rationaldesign/Sitedirectedmutagenesis
•Directedevolution(Irrationaldesign)/Random
mutagenesis
Rational design/Site directed mutagenesis
•Knowledgeofstructureandfunctionofproteinisnecessary
•Rationalgenemutationplanned
•Rationallydesignedchangesingenesofprotein–site
specificmutagenesis–proteinproductionaltered
•Advantages:Inexpensive,technicallyeasy,welldeveloped
methods
•Disadvantages:Unavailabilityofdetailedstructural
information,difficultpredictionofmutagenesis
•Structure–functionsruleapplied
•Ifproteinstructurenotavailable,directevolutionmethod
required

5. Example1:Designingof
aninactiveformof
pancreaticribonuclease
A
•From literature,
histidineatposition
119necessaryfor
catalysis
•Ifhistidinereplacedby
alanine, (H119A)
mutantofribonuclease
–nobiologicalactivity

6. Example 2: Engineering of enzyme Subtilisin
•Enzymeaddedindetergentstoincreaseefficiency
•Inactivatedbybleach
•Experimentalandstructuralanalysisprovedthatinactivation
isduetooxidationofaminoacidmethionineatposition22
(Increasedsidechainbulk,introductionstrongly
electronegativeoxygenatomnearserine–reducescatalytic
activity)
•SubtilisingeneinE.colimutatedandmethioninechangedby
alanine
•Engineeredsubtilisinwithincreasedstabilityandactivity

7. Direct Evolution/Random Mutagenesis
•Whenstructuralinformationnotavailable
•Tosolvecomplexproblem
•Randomchanges(Mutation)doneinprotein
•Largenumberofvariantscreated-Structure
–functionrulestudydone
•Mutantwithdesiredpropertiesselectedand
amplified
•Mimicsnaturalevolutionandproduce
superiorresultstorationaldesign

8. Methods
Localized or region-specific random mutagenesis:
•Combinationofrationalandrandomapproaches
•Simultaneousreplacementoffewaminoacid
residuesinaspecificregion
DNA shuffling:

9. Peptidomimetics:
•Asmallprotein-likechaindesigned
tomimicapeptide.
•Modificationofanexistingpeptide,
orbydesigningsimilarsystemsthat
mimicpeptides,suchaspeptoids
andβ-peptides.
Staggered extension process:
•Consistsofprimingthetemplate
sequence(s)followedbyrepeated
cyclesofdenaturationand
extremelyabbreviatedpolymerase-
catalyzedextension.
•Ineachcyclethegrowingfragments
annealtodifferenttemplatesbased
onsequencecomplementarity
andextendfurther.

10. Flow cytometry:
•Laserbasedbiophysicaltechnologyusedtoidentifycell
surface-displayedproteinvariantswithdesired
properties.
Cell-free translation systems:
•Celllysates(extracts)areusedforproteinexpression.
•Usedforproteinexpressionofeitherinvitro
transcribedmRNAormRNAisolatedfromtissues
orcells.
Designed diversion evolution:
•Divergentevolutionistheprocessofformationofnew
speciesaftercontinuousevolutioninancestors.
•Enzymeswithmorespecializedandactivefunctions
areevolvedbyaminoacidsubstitutions.
Stimulus-responsivepeptidesystems:
•Theabilityofpeptidesandproteinstochange
conformationsinresponsetoexternalstimulisuchas
temperature,pHandthepresenceofspecificsmall
molecules

11. Receptor-based QSAR methods:
•Structuralmodificationsleadstochangein
propertyofaprotein.
Phage-display technology:
•Inphagedisplaytechnique,ageneencodinga
proteinofinterestisinsertedintoaphage
coatproteingene,causingthephageto
displaytheproteinontheoutside.
•Mostpowerfulandwidelyusedlaboratory
techniqueforthestudyofprotein-protein,
protein-peptideandprotein-DNAinteractions.

12. Yeast surface display:
•The method of displaying
recombinantproteinsonthesurfaceofa
yeastcellviageneticfusiontoacell
wallprotein.
•Importantmethodforproteincharacterization
andidentifyingprotein–proteininteractions.

13. Applications
Industrial applications:
•Food industry:
-Proteases:Productionoflowallergenicinfant
formulas,milkclotting,flavours
-Amylases:Inbreadandbakingindustry
-Lipases:Conditioningofdoughandcheese
flavourapplications
•Detergent industry:
-Proteases:Removingproteinstains
-Amylases:Removalofstarchstains
-Lipases:Removaloflipidstains

14. Environmental applications:
•Designingofmicro-organismstoeliminate
environmentalpollutants
•Detoxificationoforganophosphorous
pesticidesandpolycyclicaromatic
hydrocarbonsbyenzymaticoxidation
Medical applications:
•Cancertreatment(Radio-immunotherapy)
•Productionoftargetedantibodies

15. Thank You…!!!