Production of Recombinant Proteins in Bacillus Subtilis

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Production of Recombinant Proteins in Bacillus Subtilis
INTRODUCTION:
Bacillussubtilis
Bacillussubtilis,isaGram-positive,catalase-positive bacterium, belongtogenusBacillus.
These are foundin the soil andthe gastrointestinaltractof ruminants andhumans.
Its alsoknows as the hay bacillusor grass bacillus.
B. subtilis is rod-shaped ,andcan form a tough,protective endospore,allowingittotolerate extreme
environmental conditions.
Size of Bacillussubtilis is1 - 1.2µm in widthand3 - 5µm inlength.
B. subtilis has historicallybeenclassifiedasan obligate aerobe,thoughevidence existsthatitis
a facultative aerobe.
B. subtilis isconsideredthe beststudiedGram-positivebacteriumandamodel organism tostudy
bacterial chromosome replicationandcell differentiation.
It isone of the bacterial championsinsecretedenzyme productionandusedonanindustrial scale by
biotechnologycompanies.
Theyare usedonplantsas a fungicide .Theyare alsousedonagricultural seeds,suchasvegetable and
soybeanseeds,asafungicide.
Bacillussubtilisbacteriaare non-pathogenic.
PROJECT AIMS
 Feasibilitystudiesof Bacillussubtilisasan expressionhostforthe productionof therapeutically
importantproteinsthroughhighcell densityfermentations.
 Studiesoninteinmediatedpurificationof therapeuticallyimportantproteinsexpressedin
Bacillussubtilis.
 Case studieswithafewproteinsof research/commercialrelevance includingmalariaproteins
that are likelyvaccinecandidates.
Motivation
1. Advantages of BacillussubtilisoverE. coli system: ProteinsexpressedinE.coli tendto form
inclusionbodies;the yieldafterrefoldingof the proteinislow.Inthe Bacillussystem,proteins

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can be secreteddirectlyintothe growthmedia.Thisfacilitatesbetterrecoveryandcheaper
downstreamprocessing.
2. Advantages of Bacillussubtilisoverfungal expressionsystems:Processtimesforsubtilis
culturesare smallerthanfora fungal system.Handlingisrelativelystraightforward,giventhatit
has beenextensivelystudiedforthe productionof industrial enzymes.
3. Native and heterologousproteinproductioninBacillus subtilis. Bacillussubtilishasthe ability
to produce enzymesinthe orderof grams perliter.Recentstudiesshoweditsabilitytoproduce
functionallyactiverecombinantproteinslike IL‐3,staphylokinase etc.,secreteddirectlyintothe
growthmedia.Therefore thisisone of the mostpromisinghostsforheterologousprotein
expression.
4. InteinMediatedProteinPurification:A rapid andeconomical purificationof recombinant
proteinsisa majorchallenge inbiotechnology.A typical recoveryprocessinvolvesanumberof
chromatographicsteps.Eachstephas to be separatelystandardizedforrecoveryof aparticular
protein.Thisistime consumingandcostly.Forlarge scale productionof proteinsof industrial or
therapeuticimportance,downstreamprocessingcostscanaccount forup to 80% of total
productioncosts.The affinitytagfusiontechnologyisafrequentlyusedalternative.A major
problemwiththe use of suchtags is inthe subsequentuse of proteasesfortagremoval.This
necessitatesadditional chromatographysteps.The use of inteinsasa fusiontechnology
simplifiesproteinrecovery.Additionof DTTor change inpH inducesself‐cleavage of inteinsand
therebyseparatesthe recombinantproteinfromthe inteintag.
5. Industrial importance of the project. Ease of handling,higheryieldsof Bacillussubtilisand
simplifyingpurificationbyusingselfsplicinginteinswouldmake adifferenceoverthe existing
methodsforheterologousproteinproduction.
Background:
1. Bacillussubtilisas an ExpressionHost
There are substantial researcheffortsongoingforthe developmentof anovel heterologousgene
expressionsystemwithsuperiorgrowthandproteinproductioncharacteristics.Productionof
heterologousproteinsathighlevelsbybacteriaiscommonlyachievedusingEscherichiacoli asthe host.
However,the E.coli expressionsystemstill hasdisadvantages.Forexample,itisa pathogenicbacterium
and heterologousproteinsmustbe endotoxinsfree.Itsecretesproteinintothe peri plasmandoften
intoinclusionbodies.Bacillussubtilisisanalternative hostforexpressionof heterologoussecretory
proteins.Advantagesof B.subtilisasanexpressionhostare:
 Huge capacity forsecretingproteinsdirectlyintothe growthmedium, whichgreatlyfacilitates
theirdownstreamprocessing.
 Widelyusedforthe productionof industrial enzymes,socultivationtechniquesare already
standardized.
 Geneticallyhighlyamenablehostorganismfromwhichalarge varietyof genetictoolshave been
developed.Several staphylococcal plasmids(pUB110and pE194) can functionin B. subtilis,and
these plasmidsformthe basisforthe developmentof variouscloningandexpressionvectors.

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 B. subtilisisnon‐pathogenic,andfree of endotoxins,andhas“generallyrecognizedassafe”
(GRAS) status.
2. INTEINS
An inteinisasegmentof a proteinthatis able toexcise itselfandrejointhe remainingportions(the
exteins)withapeptide bond.The firstinteinwasdiscoveredin1987. Since then,inteinshave been
foundinall three domainsof life (eukaryotes,bacteria,andarchaea).There are more thanapprox.200
inteinsidentifiedtodate;theirsizesrange from100‐800 aa.
Intein‐mediatedproteinsplicingoccursaftermRNA hasbeentranslatedintoaprotein.Thisprecursor
proteincontainsthree segments‐ anN‐exteinfollowedbythe inteinfollowedbyaC‐extein.Protein
splicinginvolvesthe removal of aninternal proteinsequence fromaprecursormolecule andthe ligation
of the twoflankingsequencestoproduce amature proteinproduct,ina post‐translational event
analogoustothe removal of an intronfrom mRNA.The proteinsplicingmechanismisdeterminedbythe
polypeptide sequence anddoesnotrequire otheraccessorymolecules.
Inteinshave beenusedinthe productionof industriallyimportantenzymeslike lipase,humanpituitary
adenylate cyclase,Cre recombinase etc.Theyhave alsobeenusedtoproduce therapeuticallyimportant
proteinslike GMCSF,HGH, HEGF etc. withsingle steprecoveriesbeingreported.Inall these cases,the
expression systemisE.coli.Inteinshave alsobeenengineeredforparticularapplicationssuchasprotein
synthesis,andthe selectivelabelingof proteinsegments(useful forNMRstudiesof large proteins).
Choice of Proteinsto Be Expressed
Infectiousdiseasesremainimportantcausesof morbidityandmortalityinthe developingworld.One of
the most cost effective approachestothe control of infectionsisthe developmentanddeploymentof
protective vaccines.Vaccinesmaybe formulatedasattenuatedlivingorganismsoras variousformsof
subunitvaccinesinwhichone orseveral pathogenmoleculesare presentedinassociationwithan
adjuvant.Fora numberof parasiticdiseasesinparticular,there isnoattenuatedvaccine availableeither
because of technical orsafetyissues.
For example,the bloodstagesof malariacausedbyPlasmodiumfalciparumwhichisamajorkillerof
childrencannotbe deliveredasanattenuatedvaccine because the parasite growsinside humanred
bloodcells.There wouldbe adangerthatimmunizationwithanattenuatedparasitemayraise an
autoimmune response againstthe hostredbloodcell.Accordingly,thisvaccine mustbe developedasa
recombinantproteinsubunitvaccine.Itisessential tokeepthe costof the vaccine low andthe yieldof
the proteinhighsothat it maybe widelydeployed.Thisprojectwillexamine the use of Bacillusasa
meansof large scale productionof malariaproteins.
Approach and Methodology:
I will collect soil samplesfromdifferentsitesof Pakistan.
( Peshawar.Mardan , Islamabad, Lahore, Karachi).

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AndI will analyzing toisolated Bacillusspeciesfromthatsoil samples.
The mediaI will use inthisresearchwill be nutrientbroth andagar.Bacillusspecieswill be identify by
theirphenotypicandbiochemical characteristics.
1. SuitabilityofInteinmediatedproteinpurificationin Bacillus subtilis.
 Selectionof suitable inteinandproteincombination.
 Vectorconstructionandtransformation
 Protocol developmentforinteinsplicing
 ProteinAssaystoconfirmactivity
 Comparingthe processwithtraditional (withoutinteinmediated) purificationmethod.
2. Achievinghighproductivityby means of high cell densityfermentationsvia
 Parameteroptimization(ex:pH,DO,Tempetc.)
 Mediaoptimization
 Feedingstrategy(i.e.pHstat,DO stat,exponential.) optimization
3. Scale up studies
 Studyingprocessvariationsat20 L scale
 Optimizationat20 L scale.
Experimental Planing
Malaria proteinswill be testedfortheircapacitytobe expressedinBacillussystemProteinstobe tested
include currenthighlyregardedcandidate antigensforasexualstages:‐
▬ MSP1‐19
▬ MSP4
▬ MSP5
▬ AMA‐1
These will be stringenttestsof the Bacillussystembecausetheycontainanumberof cysteinesthatform
conformational epitopesimportantforinductionof protection.
Production ofthese proteinswill assessedby:‐
 Estimationof yieldandintegrityof product
 Physico‐chemical parameterssuchasmigrationonreverse phase HPLC,Size exclusionand
anion‐exchange todetermine heterogeneityof product
 Antigenicityandevidence of formationof conformational epitopesusingaseries of monoclonal
and polyclonal antisera.
 Immunogenicity

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Proteinsfroma numberof differentspeciesof malariacouldbe tested.For example,if we produce the
proteinsfrommurine malaria,thenwe couldtestthe capacitytoprotectmice from lethal challenge and
thiscouldbe comparedto what iselicitedbythe same proteinproducedinyeastandinE.coli in the
case of MSP1‐19. Forms of the proteininP.vivax couldalsobe producedandassessed.
Work To Be Done At IIT Bombay
▬ Developaperoxide inducibleB.subtilisexpressionsystem
▬ Testthe feasibilityof intein‐mediatedproteinrecoveryinB.subtilis
▬ Standardize andscale up B. subtiliscultivationinbioreactors. Constructionof plasmidsencoding
variousantigens usingdifferentcodonbiases
▬ Assessmentof antigenicityandimmunogenicityof producedproteins
▬ Challenge model inmice if we electtoproduce some mouse malaria proteins
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