CloneaPCRamplifiedgeneinaneffortlessafternoon,andexpressrecombinantproteinthenextday.
- Five-second,directional,enzyme-freePCRcloning!90%recombinants!
- Singlecompetentcellhoststrainforbothcloningandexpression.
- TighterexpressioncontrolwithtunableRhamnose(rhaPBAD)promoter.
- Highthroughputformatfriendlywithautoinductionreagents.
AlsoavailablewithcleavableSUMOSolubilityTag
CompleteCloningandExpressionSystemswithExpressioneering™Technology
TheExpressoRhamnoseCloningandProteinExpressionSystemsaredesignedforfast,easy,andefficientdirectionalcloningandexpressionofPCR-amplifiedgenesusingExpressioneeringTechnology.ExpressioneeringTechnologyusesinvivohomologousrecombinationtoseamlesslyclonePCRamplifiedDNAintospeciallydesignedexpressionvectorswithouttheneedforenzymesorpurificationsteps.Asinglehoststrainisusedforbothstablecloningandcontrolledproteinexpression,makingExpressoRhamnosethefastestcloningandexpressionsystemsavailable.Thesystemscomecompletewithpre-processedexpressionplasmidsandcompetentcells,suppliedinsingletransformationvials.
Figure1.ExpressioneeringTechnologyusesinvivohomologousrecombinationtoseamlesslyclonePCRamplifiedDNAintospeciallydesignedexpressionvectorswithouttheneedforenzymesorpurificationsteps.Thedesiredinsertissimplyamplifiedwithprimersthatinclude18basesthatoverlapwiththeendsoftheExpresso®vector.TheunpurifiedPCRampliconisthenmixedwiththepRhamexpressionplasmidandthehigh-efficiencycompetentcellsprovided,anddirectlyplatedonappropriatemedia. |
TheExpressoRhamnoseSystemsutilizetherhamnose-inducIBLerhaPBADpromoterfortightcontrolofproteinexpression.TranscriptionfromtherhaPBADpromotercanbe“tuned”usingdifferentconcentrationsofrhamnosetoidentifytheoptimalexpressionlevelsfordifficulttargetproteins.Simpleautoinductionprotocolsusingrhamnoseandglucosesolutionssuppliedwiththekitsallowproteinexpressionwithminimalintervention.
ThepRham™N-HisandpRhamC-HisVectorsprovidedintheExpressoRhamnoseCloningandExpressionSystemfacilitateinstantcloningoftargetgeneswithachoiceofamino-orcarboxyl-terminal6xHisaffinitytags.The6xHispeptideprovidesforfastandeasyaffinitypurificationofproteinsundernativeordenaturingconditions.ForenhancedsolubleproteinexpressionusingSUMOfusiontagtechnology,seetheExpressoRhamnoseSUMOSystem.
WithinstantcloningusingExpressioneeringTechnology,asingle-hostsystemforcloningandexpression,andsimpleautoinductionprotocols,theExpressoRhamnoseSystemsarehighlyamenabletohigh-throughputcloningandproteinexpression.
Figure2.pRhamexpressionvectors.RBS,ribosomebindingsite;ATG,translationstartsite;Stop,translationendsite;Kan,kanamycinresistancegene;ROP,RepressorofPriming(forlowcopynumber);Ori,originofreplication.CloneSmart®transcriptionterminators(T)preventtranscriptionintooroutoftheinsert,andaterminatorfollowsthecloningsite.The6xHisaffinitytagisfusedtotheaminoterminus(pRhamN-His)oratthecarboxylterminus(pRhamC-His)oftheexpressedtargetprotein.Alsoavailable:pRhamN-HisSUMOVectorforenhancedsolubleproteinexpressionwithcleavableSUMOsolubilitytag.
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pRhamExpressionVectors
ThepRham™N-HisandpRhamC-HisVectors providedintheExpressoRhamnoseCloningandExpressionSystemareshowninfigure2. LikethepETiteVectorsfeaturedintheExpressoT7kits,thepRhamVectorsusedintheExpressoRhamnosekitsarebasedonthepSmartvectorbackbone,whichfeaturespatentedCloneSmart®technologyforincreasedcloningefficiency.Thepre-processedpETiteandpRhamvectorsfeaturethesamesequencesflankingthecloningsite,allowingasinglePCRproducttobeclonedintoeithervector.
Single-HostcloningandtunableexpressionwithExpressoRhamnoseSystem
Figure3.Tuningrecombinantproteinexpressionlevelswithrhamnoseinduction.ThepRhamC-HisKanVectorcontainingageneencodingabluefluorescentprotein(BFP)wastransformedintoE.cloni10Gcells.AnuninducedstarterculturewasinoculatedtoastartingOD600of0.8intoculturetubescontainingLBmediawith30µg/mlkanamycinandtheindicatedconcentrationsofrhamnose(0to0.2%w/v)or2%glucose.Afterovernightincubationat37°C,sampleswereharvestedbycentrifugation,lysedinSDS-PAGEloADIngbuffer,andanalyzedbySDS-PAGE.TheCoomassie-bluestainedgelshowstotalcellularprotein.Proteinexpressionlevelsareresponsivetorhamnoseconcentrationsbetween0.001%and0.2%.
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TherhaPBADpromoteristightlyshutoffintheabsenceofthesugarrhamnose,allowingtheuseofasinglehoststrainforbothcloningandproteinexpression.ThelevelofinductionfromrhaPBADisresponsivetodifferentconcentrationsofrhamnose(Fig.3),allowingyoutotunethelevelofexpression,especiallyforproteinsthataretoxicorinsolublewhenoverexpressed.MaximalproteinexpressionfromtherhaPBADpromoteristypicallylowerthanfromtheT7promoter,butcanstillreachveryhighlevels(upto100mg/l).
Figure4.AutoinductionofproteinexpressionwiththeExpressoRhamnoseSystem.FlasksofLBmediacontaining30 µg/mlkanamycin,0.2%rhamnose,andeither 0.05%glucose(earlyautoinduction,upperpanel)or0.15%glucose(lateautoinduction,lowerpanel)wereinoculatedtoaninitialOD600of0.4withanuninducedcultureofE.cloni10GcellsharboringtheT4ligasegeneinthepRhamN-HisVector.SamplesofthecultureswereharvestedattheindicatedtimepointsforSDS-PAGEanalysis.Inductionofligaseexpressionbeganby4hoursintheearlyautoinductionculture,or8hoursinthelateautoinductionculture.BothculturesreachedsimilarOD600by24hours. | |
Figure5.Directautoinductionofrecombinantproteinexpressionfromindividualcolonies.ThepRhamC-HisVectorcontainingtheBFPgenewastransformedintoE.cloni10GcellsandplatedonYTagarplatescontaining30µg/mlkanamycin.SinglecolonieswerepickedfromtheplateandinoculateddirectlyintoLBliquidmediacontainingkanamycin(30µg/ml),rhamnose(0.2%w/v),andeither0.05%(earlyautoinduction)or0.15%(lateautoinduction)glucose.SampleswereharvestedatthetimepointsindicatedandanalyzedbySDS-PAGE. |
ConvenientAutoinductionwithExpressoRhamnoseSystems
TranscriptionfromtherhaPBADpromoterissubjecttorepressionbyglucose.Whenbothglucoseandrhamnosearepresent,glucoseismetabolizedpreferentiallyandtherhaPBADpromoterremainsinactive.Upondepletionofglucose,therhaPBADpromoterisactivatedbyrhamnose.Convenientautoinductionprotocolsuseacombinationofglucoseandrhamnosetoallowinductionofproteinexpressionwithminimalintervention.Inoculatefromastarterculture(Fig.4)ordirectlyfromindividualcolonies(Fig.5)intoautoinductionmedia,andinductionoccursautomatically.Thetimingofinductioncanbeadjustedwiththeuseofdifferentglucoseconcentrations.SolutionsofrhamnoseandglucoseareprovidedwiththeExpressoRhamnosekits.
SeeApplicationNoteinNatureMethods,"Expresso®CloningandExpressionSystems:Expressioneering™Technologystreamlinesrecombinantproteinexpression."
Alsoavailable:ExpressoRhamnoseSUMOSystemwithcleavable6xHisSUMOtagforenhancedsolubilityofexpressedproteins.
ImportantProductUseInformation:
ThisproductisthesubjectofU.S.Patent#6,709,861.AdditionalpatentapplicationsownedbyLucigenCorporationarepending.
The6xHistagislicensedfromHoffmann-LaRoche,Inc.,Nutley,NJand/orHoffmann-LaRocheLtd.,Basel,Switzerlandandisprovidedonlyfortheuseinresearch.InformationaboutlicensesforcommercialuseisavailablefromQiagenGmbH,QIAGENStrasse1,D-40724Hilden,Germany.Purificationof6xHistaggedproteinswithNi-NTAmanufacturedbyQIAGENishighlyrecommendedforbestperformancesandtoavoidpoorpurificationresults.
SUMOExpressProteaseismanufacturedandsuppliedbyLifeSensors,Inc.
ORDERINFORMATION
TheExpressoRhamnoseCloning&ExpressionSystemcontainspre-processedpRhamN-Hisand/orpRhamC-HisVectorDNA,single-transformationE.cloni10GChemicallyCompetentCells(SOLOs),andtheauto-inductionreagents20%Rhamnosesolutionand15%Glucosesolution.AlsoincludedareN-Hisand/orC-HisPositiveControlInsertDNAs,transformationpositivecontrolpUCDNA,andforwardandreversePCRprimerstoconfirmclones.
TheExpressoRhamnoseSUMOCloningandExpressionSystemcontainspre-processedpRhamN-HisSUMOVectorDNA,single-transformationE.cloni10GChemicallyCompetentCells(SOLOs),andtheauto-inductionreagents20%Rhamnosesolutionand15%Glucosesolution.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.ebiomall.com
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最近导师要求做一个关于PARP家族蛋白在肝癌和癌旁组织中转录以及表达情况的初步研究,以确定目前的课题是否有很大价值。我之前一直在做湿实验,没有生信背景,所以对这个工作比较头疼。我不会编程,所以直接上网查找有没有可以在网上直接进行分析的网站。我一开始是上cbioportal上对肝癌的mRNA转录谱进行分析,但网站里面提供的对照是一个所谓的标准正常人。我没查到这个所谓的标准正常人是什么,而且导师说肝癌可能存在个体差异,所以要求用肝癌及其对应的癌旁组织的转录谱和表达谱进行分析。我上GEO搜索癌旁组织(paracanceroustissue),根本什么想要的结果都没有。我现在完全是一头雾水,无从下手,希望有做相关领域工作的前辈们或是做与之类似的工作的前辈们提供指导,非常感谢!
遗传物质的表达的产物是:蛋白质
DNA转录的产物是:mRNA
希望对你有帮助~
如题,我现在做出了一个蛋白相对于正常组织,在肿瘤里表达升高。想做某转录因子调控它的表达。查了转录因子预测的网站,每个预测的都很不一样,而且参与其调控的转录因子有几十个。我该怎么办呢?用什么实验技术或者方法能找到一个能做的呢?
基因表达谱测序是直接对某一物种或特定细胞在某一功能状态下产生的mRNA进行高通量测序,可以用来研究基因的表达差异情况。该技术结合了转录组测序建库的实验方法,与转录组测序相比,基因表达谱测序要求的读长更短,测序通量更小,但仅可用于基因表达差异的研究。
转录组测序是RNA水平测序,相当于DNA水平的基因组测序,是一个框架。表达谱主要研究的是基因表达量的变化,上调或下降。先要有转录组或是基因组才可以做表达谱,否则没有Ref做参考。
转录组测序和表达谱测序其实都是通过高通量测序技术进行的,转录组测序主要是针对没有参考基因组(即基因组未完成测序)的物种,侧重于获得你材料的全部转录组信息;而表达谱则侧重于检测各个基因的表达量。
转录后要进行加工,转录后的加工包括: 几乎全部的真核 mRNA 端都具“帽子”结构。虽然真核生物的mRNA的转录以嘌呤核苷酸三磷酸(pppAG或pppG)领头,但在5’端的一个核苷酸总是7-甲基鸟核苷三磷酸(m7GpppAGpNp)。mRNA 5’端的这种结构称为帽子(cap)。不同真核生物的mRNA具有不同的帽子。
mRNA的帽结构功能:①能被核糖体小亚基识别,促使mRNA和核糖体的结合;②m7Gppp结构能有效地封闭RNA 5’末端,以保护mRNA免疫5’核酸外切酶的降解,增强mRNA的稳定性。 (postranslational processing):从核糖体上释放出来的多肽需要进一步加工修饰才能形成具有生物活性的蛋白质。翻译后的肽链加工包括肽链切断,某些氨基酸的羟基化、磷酸化、乙酰化、糖基化等。真核生物在新生手肽链翻译后将甲硫氨酸裂解掉。有一类基因的翻译产物前体含有多种氨基酸顺序,可以切断为不同的蛋白质或肽,称为多蛋白质(polyprotein)。例如胰岛素(insulin)是先合成86个氨基酸的初级翻译产物,称为胰岛素原(proinsulin),胰岛素原包括A、B、C三段,经过加工,切去其中无活性的C肽段,并在A肽和B肽之间形成二硫键,这样才得到由51个氨基酸组成的有活性的胰岛素。向左转|向右转
基因表达(gene expression)是指细胞在生命过程中,把储存在DNA顺序中遗传信息经过转录和翻译,转变成具有生物活性的蛋白质分子。即表达=转录+翻译
转录量和拷贝数是相等的(产生的RNA),但和表达量(蛋白质,最终产物)不同意思,只是表达的第一步,只有转录的也都同样顺利翻译成蛋白质才有同时满足的可能。
转录增加 不等于 表达增加表达增加 也不等于 转录增加成功转录 不代表 成功表达成功表达 说明 成功转录