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.
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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.
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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
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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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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~
近期发表在《科学》(Science)以及其他杂志上的一些新研究证实，转录实际上是决定蛋白质丰度中最具影响力的步骤。
例如，近期来自哈佛-麻省理工Broad研究所的Marko Jovanovic等在Science杂志上发表文章，称检测了处于稳定状态和响应细菌脂多糖(LPS)时的小鼠骨髓树突细胞。他们发现在稳定状态时，mRNA水平、翻译速度和蛋白质降解速度可分别解释68%、26%和8%的蛋白质表达差异。当用LPS刺激细胞时，mRNA水平似乎可以解释90%的蛋白质表达差异，而翻译和蛋白质降解只能解释4%和6%的差异。Jovanovic等发现，在LPS处理的情况下，核糖体、线粒体以及其他一些高表达管家蛋白的翻译和蛋白质降解速度发生了更多改变，表明这两个步骤在控制一些过程中发挥了重要的作用。
来自加州大学洛杉矶分校统计学和人类遗传学助理教授Jingyi Jessica Li，和劳伦斯伯克利国家实验室的Mark Biggin，则在PeerJ杂志的一篇论文中用两种方法重新分析了2011年一项Nature研究的数据，说明了一些检测错误。他们证实采用第一种方法结果表明mRNA水平差异可以解释最小56%的蛋白质水平差异，而第二种方法表明mRNA水平可以解释84%的蛋白质表达差异，其中转录占73%，RNA降解占11%，而翻译和蛋白质降解各自仅占8%。
在3月6日，发表在Science杂志上的一篇题为“Statistics requantitates the central dogma”的文章中，Li和Biggin综述了上述这些近期的研究，得出了转录是蛋白质丰度最大贡献者这一结论。他们认为，他们自身以及近期其他一些研究工作都采用了更细致的统计学方法，来评估或是减少了实验性检测错误。Li和Biggin认为，早期的一些研究得到的有关翻译影响的结果实际上是由于实验错误所导致。
研究人员提出，科学家们精确地模拟基因表达需要采用更准确的测量和分析方法。他们的研究对于鉴别出可以有效治疗各种疾病的药物具有重要的影响。
原文链接：Statistics requantitates the central dogma
Dynamic profiling of the protein life cycle in response to pathogens
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？
1.基因丢失：体细胞分化过程必须将某些基因永久性的关闭,最简单有效的方式就是将其丢失.2.基因扩增：发育分化、环境条件的改变,对某些产物的需要量急剧增加--增加该基因的拷贝数.3、基因重排：某些基因片段改变原来的书顺序重新排列.4、甲基化修饰,脊椎动物,DNA上特定的CpG序列的C处可发生甲基化修饰.5、染色质结构的修饰.
检测目的基因是否转录通常采用分子杂交的方法。 基因表达分为转录及翻译两阶段，转录是以DNA（基因）为模板生成mRNA的过程，翻译是以mRNA为模板生成蛋白质的过程，检测外源基因的表达就是检测特异mRNA及特异蛋白质的生成。
请教各位，我是做在体实验，在体给予刺激30分钟后观察蛋白的表达变化，结果是蛋白表达升高，但有人质疑30分钟这么短的时间能否真的引起蛋白表达的明显变化，所以请教各位，一般蛋白从转录到表达需要多长时间？
转录是遗传信息由DNA转换到RNA的过程。作为蛋白质生物合成的第一步，转录是mRNA以及非编码RNA（tRNA、rRNA等）的合成步骤。是遗传信息从DNA流向RNA的过程。即以双链DNA中的确定的一条链（模板链用于转录，编码链不用于转录）为模板，以ATP、CTP、GTP、UTP四种核苷三磷酸为原料，在RNA聚合酶催化下合成RNA的过程。
