- Savetime!Enzyme-freecloninginsecondswithcloning-readyvectorandcells.
- Highefficiency:›90%recombinants.
- Tightly-controlledexpressionofN-orC-terminal6xHistaggedproteins.
AlsoavailablewithcleavableSUMOSolubilityTag
CompleteCloningandExpressionSystem
TheExpressoT7CloningandProteinExpressionSystemsaredesignedforfast,easy,andefficientdirectionalcloningandexpressionofPCR-amplifiedgenes.Thesystemsarecompletewithpre-processedpETite™N-orC-Hiscloningvectors,andtwocompetentcelllines,suppliedinsingletransformationvials.HighefficiencyHI-Control™10GChemicallyCompetentCellsenablestablecloningandHI-ControlBL21(DE3)CompetentCellsprovidetightlycontrolledproteinexpression,thushelpingyouavoidexpressionproblemsseenwithleakyT7promoter-basedsystems.TheN-orC-terminal6xHistaggedproteinscanberapidlypurifiedusingstandardNichromatography.SystemsarealsoavailablethatencodeanN-terminal6xHisSUMOtagfusionforimprovedsolubilityofexpressedprotein.OtherExpressoSystemsareavailablewhichexpressproteinsunderthecontroloftheRhamnosepromoter.
Five-SecondDirectionalCloningofPCR-AmplifiedGenes
TheExpressoT7CloningandProteinExpressionSystemusesExpressioneeringTechnology™,anenzyme-freerecombinationalcloningstrategytoseamlesslyintegratethegenewiththevector.ThetargetgeneisamplifiedbyPCRusingprimersthatadd18base-pairsofvector-complementarysequencetobothendsofthegene.Unlikeotherrestrictionenzymebasedmethodsorligase-freecloningmethods,nofurthercleanuporenzymatictreatmentofthePCRproductisnecessary.Simplymix1µloftheunpurifiedPCRreactionwiththesuppliedpre-processedpETite™T7expressionvector,andtransformimmediatelyintotheHI-Control™10GChemicallyCompetentCellsprovided(Figure1).
Cloning-ReadyVectorsSaveHoursofResearchTime.
NewpETiteT7vectorsinclude:
- StrongT7promoterforhigh-levelexpression.
- ChoiceofN-terminalorC-terminal6xHisfusiontagsforfastproteinpurification.
- Smallsize(2.2kb)foreasierdownstreammanipulation.
- PatentedCloneSmart®technologyincreasescloningefficiency.
Figure2.pETiteT7expressionvectors:Smallsize(2.2kbvs.5.4kbforpET)foreasiermanipulation,includingtargetedmutagenesis.Vectorsarepre-processedforinstantenzyme-freecloningofPCRproductswithachoiceofamino-terminalorcarboxyl-terminalfusionof6xHistagtoproteinofinterest. |
HighcloningefficiencywithHI-Control10GChemicallyCompetentCells.
ThehightransformationefficiencyofHI-Control10GChemicallyCompetentCellsensuresrecoveryofcloneswithprecisejunctionsandthecorrectorientation.Formostgenes,>90%ofcolonieswillhavethetargetgeneinsertedinthecorrectorientation.(Figure3).
Figure3.HighcloningefficiencywithExpressoT7System. |
HI-ControlBL21(DE3)CellsControlLeakyProteinExpression
HI-ControlBL21(DE3)CellscontainhighlevelsoflacrepressortomaintaintightcontroloverexpressionofT7RNApolymerase.Tightercontrolmeansbettertoleranceofpotentiallytoxicgeneproducts(Figure4).
Figure4.ExpressionofvariousproteinsusingtheExpressoT7SystemversusapETVector.GenesencodingaDNApolymerase,abluefluorescentprotein(BFP),orATPsynthasebsubunit(membraneprotein)wereclonedintopET28aorpETitevectorswithN-terminalorC-terminal6xHistags(asindicated).pET28aclonesweretransformedintostandardBL21(DE3)cells,andpETiteclonesweretransformedintoHI-ControlBL21(DE3)cellsforexpression.CulturesweregrowninLBat37°CtoanOD600of0.5to0.7(odd-numberedlanes)andinducedfor3hourswith1mMIPTG(even-numberedlanes).CellswerepelletedandlyseddirectlyinSDS-PAGEloADIngbuffer,and0.05ODequivalentswereanalyzedbygelelectrophoresis.ThegelwasstainedwithCoomassieblue. |
PurificationofActiveSolubleFluorescentProteinUsing6xHisTag
TheN-orC-terminal6xHistaggedproteinsexpressedusingtheExpressoT7CloningandExpressionSystemcanberapidlyaffinity-purifiedovercommerciallyavailablenickelresins.Anexampleofthepurificationofa6xHistaggedyellowfluorescentproteinclonedandexpressedusingtheExpressoT7SystemisshowninFigure5.
Figure5.Purificationofa6xHistaggedfluorescentprotein.HI-ControlBL21(DE3)cellsharboringpETiteC-Hisvectorcontainingayellowfluorescentprotein(YFP)geneweregrownat37°CinLBmediatoanOD600of0.6(lane1),theninducedwith |
ImportantProductUseInformation:
ThisproductisthesubjectofU.S.Patent#6,709,861.AdditionalpatentapplicationsownedbyLucigenCorporationarepending.
The6xHistagislicensedfromHoffmann-LaRoche,Inc.,Nutley,NJand/orHoffmann-LaRocheLtd.,Basel,Switzerlandandisprovidedonlyfortheuseinresearch. InformationaboutlicensesforcommercialuseisavailablefromQiagenGmbH,QIAGENStrasse1,D-40724Hilden,Germany.Purificationofthe6xHistaggedproteinswithNi-NTAmanufacturedbyQIAGENishighlyrecommendedforbestperformancesandtoavoidpoorpurificationresults.
ORDERINFORMATION
TheExpressoT7Cloning&ExpressionSystemcontainspre-processedpETite®N-Hisand/orpETiteC-HisVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHI-ControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareN-Hisand/orC-HisPositiveControlInsertDNAs,andtransformationpositivecontrolpUCDNA.TheExpressoT7SUMOCloningandExpressionSystemcontainspre-processedpETite®N-HisSUMOVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHIControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.
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但严格来说,还是有区别的。因为基因的表达产物包括蛋白质和RNA(rRNA和tRNA)。如果产物是蛋白质,就是转录翻译;如果产物是RNA,就只是转录过程,没有翻译过程。
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
应用高通量技术进行转录组测序是一种快捷可靠的获取转录组信息的方法。mRNA的转录本表达分析,通过获得研究对象基因组转录区域的信息,鉴定转录发生位点,可变剪切等,其精确的计数方法更可对基因进行精确的定量分析。
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?
翻译是指mRNA在核糖体的帮助下翻译成肽链
基因表达则是二者的统一,即DNA转录成RNA,RNA翻译成肽链,并最终折叠成有意义的蛋白质
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
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