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).
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 |
|
Activatedcarbon |
|
Surfactants |
|
Anion-exchangechromatography |
|
Histamine-andhistidine-immobilizedSepharose |
|
PolymyxinB-immobilizedSepharose |
|
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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.
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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。
各位,最近几个月一直在帮着公司进口细胞株,我是新手,从来没有接触过这样的业务,摸着石头过河,遇到了很多问题,也得到了很多教训和经验,不敢独享,觉得还是分享一下吧,给有需要的指指道,希望能少走弯路。
先不多说,正文如下:
大家看好了,我写的是企业进口细胞株,不是针对高校或者别的什么科研机构,因为到目前为止国家还没有一个正式的文件去指导企业进口细胞株,只有一个过渡性文件,因此各地操作时也有些不同,除了北京、上海、广东等一些发达的地方有操作程序,其他地方由于企业进口细胞株的案例很少,因此很麻烦的(比如黑龙江,我们最后都找到省卫生厅厅长了)。
首先,你们公司如果选择购买一个国外的细胞株,最先考虑的是付款问题,你得找一家进出口代理公司,帮你操作,这个都是比较常规的,另外你最好再找一家在北京或者上海的公司帮助清关。这里有涉及到“进口代理协议”和“供货合同,我们当时是签了一个三方合同”,这里一定要注意和国外沟通好,需要进口的细胞株的名称、数量、价值等等,后期发货的时候千万不要不一致。
因为细胞株是特殊物品,需要从省卫生厅那里拿到一个“医用特殊物品准出入境证明”,这个东西很难搞的(高校或科研机构容易一些,基本上都有程序),尤其是一些没有先例的省份(领导不敢批,怕担事),我们公司就是黑龙江的先例,这个东西不知道怎么办的到时候可以找我私聊。搞这个东西的正常周期是2-3周,请注意,一定要把准出入境的有效时间尽量写长一点,还有就是主送单位一定要你所清关的口岸的检疫局,例如北京口岸入境,就写北京出入境检验检疫局。当时没人提醒我,这两个我都搞错过,结果多跑了几次,我们的国家服务人员态度一次比一次差,快气死了,不过后来还是搞到了。
拿到“医用特殊物品准出入境证明”以后,预付款也打了,国外准备要发货了,这之前还要一个“特殊物品入境审批单”,这个需要到你所清关的口岸的检疫局搞,下面以北京出入境检验检疫局为例,这个东西要是你是外地的(非清关所在口岸)是搞不到的,你得先去北京出入境检验检疫局备案(就是公司证照原件,情况说明,单位说明等等,比较好办),然后在你细胞株进口之前2-3周申请(1周能批,有效期3个月)。我当时是委托北京一家公司办的,因为你自己办是不方便的,到时候审批单还要亲自去取,外地的很麻烦。
箱单、发票就不说了
接下来是运输,细胞株一般都用干冰运输,当然也可以选择液氮,不过价钱不一样,液氮要比干冰贵40%左右。要计算好运输过程中干冰损失量,量要足够,最好放一个温度记录仪,如果你没有把握,在货物到港清关之前,也可以加干冰的,就比较贵,单次1000元左右。发货之前一定和发货方、运输公司沟通好,标签和数量一定要准入证明和审批单一致,要不然清关很麻烦的,我再次犯了一个错误。
最后就是清关了,这里一定要注意,你找的清关公司的清关能力一定强,一定是做过冷链清关的。
总结一下:
程序是:签合同,找进口代理和清关公司,办理相关证明文件,选择冷了运输公司,运输,清关。
文件:合同、代理协议、医用特殊物品准出入境证明、特殊物品入境审批单、发票、箱单等。
另外,这里面还有不少细节,我就不一一说了,如果真有这方面运输的,可以找我。
版主horizon801留言:
谢谢分享
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?
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
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.