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).
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| 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).
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| 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 |
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Activatedcarbon |
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Surfactants |
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Anion-exchangechromatography |
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Histamine-andhistidine-immobilizedSepharose |
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PolymyxinB-immobilizedSepharose |
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ProteinExpressionwithClearColi
TherelativeproductionefficiencyofendotoxinfreeClearColi™BL21(DE3)competentcellswascomparedtonormalBL21(DE3)cellsusingtwodifferentrecombinantproteins. AlthoughoverallgrowthratesoftheClearColiareslower,finalproteinproductionlevelsareverysimilar(seeFig.3and4).
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| Figure3.ComparisonofproteinexpressioninClearColiBL21(DE3)andLucigen'sE.Cloni®EXPRESSBL21(DE3)competentcells.CellscontainingaT7expressionplasmidharboringageneencodingthehumanapolipoproteinA1(ApoA1)weregrowninLBMillermediumat37°C.WhenculturesreachedOD600of0.6to0.8,expressionwasinducedbytheadditionof0.4mMIPTGandincubationwascontinuedfor3hours.Equivalentnumbersofuninduced(-)andinduced(+)cellswerelysedbyheatinginLaemmlibufferandsampleswereanalyzedbySDS-PAGEona4%-20%polyacrylamidegrADIentgel. |
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| 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).
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| 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.
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| 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.
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| 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.
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如题，求助。做肝癌细胞侵袭、迁移实验，用哪种细胞株比较好？
病情描述(发病时间、主要症状等)：
体检血常规中血小板计数343.血小板比积0.39.
尿检中白细胞计数26.2.上皮细胞计数10.30
心电图下写窦性心律，p-r间期偏短
想得到怎样的帮助：
有没有什么大问题
你的第一部分，白细胞和中性粒细胞偏低，说明免疫力低，最近要多保养，不受凉，近期容易生病。不过数据也不是太低的，也不用过于担心，多吃多睡，过一段时间复查，可能会好转为正常。另外消炎药，止痛，退热药尽量不吃，这些药会加重白细胞下降的。
第二部分，最主要的红细胞和血红蛋白是正常的，总体看还是正常的。但是下面数据有点低，而且只低一点，问题不大，多吃点米饭，红枣，肉等，会好转的。
第三部分，血小板部分是正常的。
你这种检查单，最主要的是免疫力低下，注意多吃多睡，别受凉，少烦神少劳累，引起白细胞低的药物尽量不吃。
G418浓度之前师兄已经摸索出来，这次筛选直接用师兄浓度。
1、6孔板转染后多长时间开始进行G418筛选？
2、G418筛选是在6孔板中继续筛选还是转到24/96孔？大皿？
3、G418浓度什么时候减量
4、？筛选过程中荧光观察一小部分克隆不错，如何挑选出来？
5、整个筛选过程大约需要多长时间？
lipo3000筛选
谢谢大家！
目前想建立家系血标本的淋巴细胞的永生化细胞株，请问有哪些成熟的方法及protol，费用贵么？
希望有大神赐教!谢谢！
