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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红细胞压积，是指红细胞占血液体积的比例，您仅仅是比参考范围略低，同时红细胞数量，和血红蛋白并没有下降，这可能是血液有所稀释所致。
白细胞计数3.9，正常的话 4-10之间，你仅仅是比参考范围略下降一丁点，本身并无意义，参考范围是大致的。而且血液还可能存在稀释可能性
中性粒细胞绝对值和百分比略低一点，同时淋巴细胞百分比略有上升，那这本身就只是略高一点点，而一般比如感冒以后，也往往可以升高，其他的红细胞分布宽度等是派生指标，没大意义的。
总之单单看这一个血常规检验，本身没有什么大的异常发现，当然也可以稍后复查，比如过2周以后复查
我是厉清，更多医疗知识，请看 厉清就是我 的新浪博客
单纯从你这个尿常规结果来看：你的上皮细胞数量在正常女性的尿液中算是正常的，镜检白细胞：15——20个/HP，它表示你的泌尿系统可能存在感染。但是，因为没有你的其他资料和其他检查结果，单凭一个结果不能说明太多问题。一般情况下不可能是肾的不好，请不用担心。
我的建议是：
1、去医院复查，但要注意留尿的时候先清洁外阴，留取中段尿。及时化验。
2、如果检查结果仍然有白细胞，可去妇科或泌尿科看医生，一般的感染的话吃点药马上就好了。
3、放松心情，注意个人卫生即可。
生物工程包括五大工程，即遗传工程(基因工程)、细胞工程、微生物工程(发酵工程)、酶工程(生化工程)和生物反应器工程。在这五大领域中，前两者作用是将常规菌(或动植物细胞株)作为特定遗传物质受体，使它们获得外来基因，成为能表达超远缘性状的新物种——“工程菌”或“工程细胞株”。后三者的作用则是这一有巨大潜在价值的新物种创造良好的生长与繁殖条件，进行大规模的培养，以充分发挥其内在潜力，为人们提供巨大的经济效益 和社会效益。
生物工程的应用领域非常广泛，包括农业、工业、医学、药物学、能源、环保、冶金、化工原料等。它必将对人类社会的政治、经济、军事和生活等方面产生巨大的影响，为世界面临的资源、环境和人类健康等问题的解决提供美好的前景。展开
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？
看到之前的一个朋友也发了IBC会议的PPT内容，不过好像没有这个PPT（不确定，要是重复了请见谅）。
因为会议上投影的问题，当时有些内容贾老师没介绍太多。不过内容相当不错，主要是国内比较缺乏这方面的信息和经验。可以算得上是干货吧，不过里面主要是一些规则性或方向性的内容，需要具体细节，只能花钱请她做顾问了。
AudreyJia是美籍华人，在FDA做CMCReviewer之前也一直是从事细胞开发及细胞培养的工作。她应该也是目前唯一一个生物大分子部门中，唯一从FDA离职出来从事顾问工作的。可能很多国内单抗药企大佬都认识她，可能很多园子里面的人也见过她了。
谁能赏几个积分啊？新人，没积分，一些东西想看或者下载都不行。
版主jackieustc留言:
感谢分享，鼓励新人，我给你打赏，多交流自然有收获！
Monoclonality_JIA1.pdf(952.57k)
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。
