Eliminateendotoxinatthesource

  • GeneticallymodifiedLPSdoesnottriggerendotoxicresponseinhumancells
  • Idealformammaliancellimmunogenicitytesting,toxicityassays,andtherapeuticproteindrugdiscovery
  • Usefulformembraneandlipidbindingproteinproduction
  • ProteinexpressionsimilartoBL21(DE3)cellswithoutrequiringdownstreamendotoxinremoval
  • Reducefalsepositivesincytokineassays,improveconfidenceinyourresults
  • FrequentlyAskedQuestions

  • Introduction
  • Genotypeinformation
  • Whyendotoxinremovalisnotthebestmethod
  • ProteinexpressionwithClearColi
  • Endotoxicityassayresults
  • LALtesting:doesitreallymeasureendotoxin?
  • GrowthratesforClearColiBL21(DE3)cells
  • Usefulreferencearticles
  • ClearColilicensinginformation

IntroductiontoClearColi®technology:

Isthereabetterwaytoeliminateendotoxincontamination?
Nowthereis. 

Insteadofremovinglipopolysaccharide(LPS)contaminationfromyourproteinorplasmidDNApreparations,eliminatetheLPSatthesource.  GeneticallymodifiedLPSfromanovelE.colistrainproducesfunctionallycleanrecombinantproteinsandplasmids. ClearColi®BL21(DE3)cellsarethefirstcommerciallyavailablecompetentcellswithamodifiedLPS(LipidIVA-seeFig.1)thatdoesnottriggertheendotoxicresponseinmammaliancells.ClearColi™cellslackoutermembraneagoNISTsforhTLR4/MD-2activation;therefore,activationofhTLR4/MD-2signallingbyClearColiisseveralordersofmagnitudelowercomparedwithE.coliwild-typecells,andheterologousproteinspreparedfromClearColiarevirtuallyfreeofendotoxicactivity.AfterminimalpurificationfromClearColicells,proteinsorplasmids,whichmaystillcontainLipidIVA,canstillbeusedinmostapplicationswithoutelicitinganendotoxicresponse(seeEndotoxicityassayresultsfordetails).

ClearColi System
Figure1.TheLPSofanormalE.colicellcomparedtothegeneticallymodifiedLipidIVAfromClearColicells. InClearColi,theoligosaccharidechainhasbeendeleted,andtwoofthesixacylchainshavebeenremovedtodisabletheendotoxinsignal.

ModificationstothegenotypeoftheClearColicellsconsistofsevenseparategenedeletions,therebyensuringthereisnochanceofgeneticreversionbacktowildtypeandproductionofnormalLPS. ThesemutationsresultinthedeletionoftheoligosaccharidechainfromtheLPS,makingiteasiertoremovetheresultinglipidIVAfromthedownstreamproduct. Moreimportantly,twoofthesixacylchainsaredeleted. ThesixacylchainsoftheLPSarethetriggerwhichisrecognizedbytheToll-likereceptor4(TLR4)incomplexwithmyeloiddifferentiationfactor2(MD-2),causingactivationofNF-ƙBandproductionofproinflammatorycytokines. LipidIVA,whichcontainsonlyfouracylchains,isnotrecognizedbyTLR4andthusdoesnottriggertheendotoxicresponse(seeFig.2).

Fig.2.ComparisonofrelativeNF-κBinductioninHEK-BlueCellsusingpurifiedLPSfromaK-12E.colistrainorfrompure,syntheticallymanufacturedLipidIVA.

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GenotypeInformation:

ClearColiBL21(DE3)competentcellshavethefollowinggenotype:
F–ompThsdSB(rB-mB-)galdcmlon&lamBDa;(DE3[lacIlacUV5-T7gene1ind1sam7nin5])msbA148ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA

Sevenspecificdeletionmutations(ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA)encodethemodificationofLPStoLipidIVA,whileoneadditionalcompensatingmutation(msbA148)enablesthecellstomaintainviABIlityinthepresenceoftheLPSprecursorlipidIVA.

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WhyEndotoxinRemovalisnottheBestMethod

Lipopolysaccharide(LPS),alsoknownasendotoxin,isapotentagonistforhTLR4/MD-2-mediatedproinflammatoryactivityinhumanimmunecells.Topreventtoxicity,recombinantproteins,commonlymanufacturedinE.coli,mustbeessentiallyfreeofendotoxin.However,efficienteliminationofendotoxinisachallengingtask,andnoneofthedownstreamapplicationsremoveendotoxinentirely.Currentmethodsforendotoxinremovalfromrecombinantproteinsarevaried,includingultra-filtration,activatedcarbon,surfactants,anionexchangechromatography,andimmobilizedsepharose. Eachofthesestrategiesinvolvesnegativeeffects:significantyieldloss,highcost,lossofbioactivityoftheprotein,orbioactivityoftheadditivesusedforendotoxincleanup. Table1belowdescribessomeofthemostcommonmethodsandtheirassociatedshortcomings:

Table1: 

 Method

Disadvantages

Ultrafiltration

  • Onlyusefulforsmallproteins
  • Endotoxinmonomersmaypermeatethemembraneduetoendotoxin/proteininteraction
  • Inefficientforproteinswhichcanbedamagedbyphysicalforces

Activatedcarbon

  • Adsorbingactivityforbothendotoxinandprotein

Surfactants

  • Expensive
  • Mayaffectbioactivityofprotein
  • Difficulttocompletelyremove
  • Removalmayleadtoproductloss

Anion-exchangechromatography

  • Highadsorbtionofbothendotoxinandacidicprotein
  • Noselectivitytoadsorbendotoxin

Histamine-andhistidine-immobilizedSepharose

  • Removingcapacitydependentontheionicstrength
  • BIOLOGicalactivityofhistamine

PolymyxinB-immobilizedSepharose

  • ProteinlossesduetotheionicinteractionbetweenpolymyxinBandprotein
  • PolymixinBisphysiologicallyactive

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ProteinExpressionwithClearColi

TherelativeproductionefficiencyofendotoxinfreeClearColi™BL21(DE3)competentcellswascomparedtonormalBL21(DE3)cellsusingtwodifferentrecombinantproteins. AlthoughoverallgrowthratesoftheClearColiareslower,finalproteinproductionlevelsareverysimilar(seeFig.3and4).

Figure3.ComparisonofproteinexpressioninClearColiBL21(DE3)andLucigen'sE.Cloni®EXPRESSBL21(DE3)competentcells.CellscontainingaT7expressionplasmidharboringageneencodingthehumanapolipoproteinA1(ApoA1)weregrowninLBMillermediumat37°C.WhenculturesreachedOD600of0.6to0.8,expressionwasinducedbytheadditionof0.4mMIPTGandincubationwascontinuedfor3hours.Equivalentnumbersofuninduced(-)andinduced(+)cellswerelysedbyheatinginLaemmlibufferandsampleswereanalyzedbySDS-PAGEona4%-20%polyacrylamidegrADIentgel.
Figure4.ComparisonoffluorescentproteinexpressioninClearColiBL21(DE3)andE.cloniEXPRESSBL21(DE3)cells. ApETexpressionvectorharboringageneencodingayellowfluorescentprotein(LucY)underthecontrolofaT7promoterwastransformedintobothClearColiBL21(DE3)andE.cloniEXPRESSBL21(DE3).ColonieswereinoculatedintoLB-Millermediumandgrownat37°Cforinduction.Samplesofinducedanduninducedcellscontainingequivalentnumbersofcellswerecentrifuged,andcellpelletswerephotographedunderlong-wavelengthUVIllumination.

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EndotoxicityAssayResults

Inmostcases,asimplenickelcolumnpurificationissufficientforendotoxicshockresponsenegativerecombinantproteinssuitableordownstreameukaryoticcellularresponse assays. ResearcherscannowassaytargetproteineffectswithoutconcernthatimmuneresponsetriggersarearesultofLPSendotoxincontamination. Todemonstrate,ApoA1proteinexpressedfromClearColiBL21(DE3)competentcellswasNi-columnpurifiedandtestedforendotoxicityusingboththeHEK-BluecelllinefromInvivoGen(seeFig5) andtheLALassay(seeFig.6).

Figure5.ComparisonofendotoxicresponsefromproteinderivedfromClearColiBL21(DE3)andtraditionalBL21(DE3)competentcells 

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LALTesting:Doesitreallymeasureendotoxin?

LimulusamebocyteassaytestingisanFDA-approvedmethodfordetectionofendotoxinsandthemostcommonassayused;howevertheLALassayisactivatedsolelybythe4´-monophosphoryldiglucosaminebackboneofLPS. LALactivityisminimallyinfluencedbyacylationpatternofLPS,thekeydeterminantofendotoxicityineukaryoticcells. TheLALassayalsorecognizesawiderspectrumofLPS/lipidAvariantsthanthecentralcellularendotoxinsensorsystemofthehumanimmunecellsystem. Assuch,falsepositiveresultscanandwillresultduetothelackofspecificityoftheassay.

AsimpleNi-columnpurificationstepforproteinsproducedfromClearColicellswillreduceLALresponselevelsby95%orgreater(seeFig.6). However,theresidualEUmeasurementsareduetothenon-specificnatureoftheassayunlessextraneousLPScontaminationfromothersourcesispresent. Alternativetoxicityassays,suchasthoseusingHEK-Bluecells(seeFig.5)suggestthateveninthepresenceofEUlevelsabovethreshholdsnormallytargetedbyresearchers,theactualimmunogeniceffectsfromClearColi-derivedproteinsarenon-existent.

Duetothenon-specificityoftheLALassaywhencombinedwithlipidIVAfromClearColi,itissuggestedthatresearchersconsideralternativemethodsofendotoxinmeasurement.

Figure6.Comparisonof endotoxinunitsasmeasuredbytheLALassayusingnickel-columnpurifiedrecombinantApoA1andHSPproteinexpressedinClearColiBL21(DE3)(redbars)andE.cloniEXPRESSBL21(DE3)(greybars)competentcells. ProteinexpressedfromClearColidemonstratessignificantreductioninEU/mgwithoutendotoxinremovalsteps.

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GrowthRatesforClearColiBL21(DE3)Cells

ClearColiBL21(DE3)cellsgrowatapproximately50%oftherateofnormalBL21(DE3)cells(seeFig.7). Usersshouldexpecttoseeverysmallcoloniesforthefirst24hoursafterplatingtransformants. Lucigenrecommendsincubatingplatesfor32-40hoursbeforepickingcoloniesforfutureexperiments. Whengrowntosufficientdensities,ClearColiBL21(DE3)cellsproducesimilarproteinlevelsasnormalBL21(DE3)cellswhencomparingequalnumbersofcells.

Figure7.ComparisonofgrowthratesforClearColiBL21(DE3)vs.E.cloniEXPRESSBL21(DE3)cells.CellswereinoculatedtoaninitialOD600of~0.003in200mlofLBMillermediumandgrownat37°Cwithshakingat210rpm.TheOD600 ofthecultureswasrecordedhourly.

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RelevantReferenceArticles:

  • Teghanemt,etal,MolecularBasisofReducedPotencyofUnderacylatedEndotoxins,JImmunol,2005;175:4669-4676
  • Mamat,etal,SingleaminoacidsubstitutionsineitherYhjDorMsbAconferviabilityto3-deoxy-D-manno-oct-2-ulosonicacid-depletedEscherichiacoli,MolecularMicrobiology,2008,67(3),633–648
  • Meredith,etal,RedefiningtheRequisiteLipopolysaccharideStructureinEscherichiacoli,ACSChemicalBiology,2006,1(1),33-42
  • Brandenburg,etal,TheExpressionofEndotoxicActivityintheLimulusTestasComparedtoCytokineProductioninImmuneCells,CurrentMedicinalChemistry,2009,16,2653-2660
  • Gutsmann,etal.StructuralprerequisitesforendotoxicactivityintheLimulustestascomparedtocytokineproductioninmononuclearcells,InnateImmunity,2010,16(1),39-47
  • BeomSeokPark1etal.,ThestructuralbasisoflipopolysacchariderecognitionbytheTLR4–MD-2complex,Nature458,1191-1195(30April2009)

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ClearColiLicensingInformation:

ClearColi™CompetentcellsaresubjecttoUSPatent8,303,964andotherUSandforeignpendingpatents.

LucigenCorporation("Lucigen")hasalicensefromResearchCorporationTechnologiestosellClearColicompetentcellstothird-partiesfornon-commercialresearchpurposesonly.Aseparatelicenseisrequiredforanycommercialuse.Formoreinformationabouttheuseofthisproductbycommercialentities,pleasereviewour fulllicensingpage.

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ORDERINFORMATION

Each ClearColi®BL21(DE3)ElectocompetentCellKitcontains:ClearColiBL21(DE3)ElectocompetentCellsinDUOpackaging(2transformationspertube),ExpressionRecoveryMedium(lactoseminus),pUC19PositiveControlPlasmid,andcompleteprotocols.

ExpressionRecoveryMedium(lactoseminus)isalsoavailableseparately.