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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希望有大神赐教!谢谢！
指导意见：
毕竟阴道上皮是鳞状上皮,而输尿管,膀胱,尿道的上皮是柱状上皮,形态上是不一样的.所以您要是对您的结果出怀疑态度,首先：重新留尿,做一个复检（留尿时注意防止阴道分泌物污染）；或者让值班医生分析一下是那种类型的上皮（鳞状还是柱状）.所以不要担心这个结果.一般情况下污染的比较多.
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
如题，求助。做肝癌细胞侵袭、迁移实验，用哪种细胞株比较好？
2：红细胞计数（RBC）（参考值：3.5~5.5），（单位：10^12/L）
3：血红蛋白浓度（HB）（参考值：120~160），（单位：g/L）
4：红细胞压积（HCT）（参考值：40~48），（单位：%）
5：平均红细胞体积（MCV）（参考值：80~97），（单位：fL）
6：平均红细胞血红蛋白含量（MCH）（参考值：26.5~33.5），（单位：pg）
7：平均红细胞血红蛋白浓度（MCHC）（参考值：300~360），（单位：g/L）
8：血小板计数（PLT）（参考值：100~300），（单位：10^9/L）
9：淋巴细胞比值（LY%）（参考值：17~48），（单位：%）
10：单核细胞比例（MONO%）（参考值：4-10），（单位：%）
11：中性粒细胞比例（NEUT%）（参考值：43~76），（单位：%）
12：淋巴细胞计数（LY）（参考值：0.8~4.0），（单位： 10^9/L）
13：单核细胞计数（MONO）（参考值：0.3~0.8），（单位：10^9/L）
14：中性粒细胞计数（NEUT）（参考值：1.2~6.8），（单位：10^9/L）
15：红细胞分布宽度（参考值：11~14.5），（单位：%）
16：血小板体积分布宽度（PDW）（参考值：9~18），（单位：%）
17：平均血小板体积（MPV）（参考值：7．4~12．5），（单位：fL）
18：大血小板比例（P-LCR）（参考值：10~50），（单位：%）
