信號(hào)肽酶切割

1、HowdaIclonemygeneatinterestintopPICGaorpPICZatoensurethatProductFAQtherearenoextraaminoacidsattheN-terminusafterthealphasecretionsignaliscleavedbyKex2?AnswerKex2cleavesthealphas&cretionsignalpeptidefromtheN-terminusoftheover-expressedproteininyeast.WhileSte13generallyremovestheGlu-Alaaminoacidswhich

2、arebetweentheKEX2siteandyourexperimentalproteintheSte13cleavagemaynotlecomplete.ToensurethattheN-terminusofyourproteinisexposedafterKex2cleavage,youmaycloneyourgeneofinterestflushwiththeKex2cleavagesite.InordertocloneyourgeneflushwiththeKEX2cleavagesite,youmustcutthepPICavectorwithXhol(andanyotherap

3、propriateenzymeforcloninginframewith3endofthevector).BycuttingthevectorwithXholalone,youwillexcise62-67nucleotidesofthev&ctor(dependingonwhichv&ctoryou日rmusing)includirigtheKEX2signalcleavagesite.Inordertoregeneratethissignalcleavagesite,youwillneedtogenerateaforwardprimerwhichcontainstheXholsite,fo

4、llowedbytheKEX2cleavagesequenceandnucleotidesfromyourgeneofinterestAfteramplificationofyourgene,youwilldigestyourPCRproductwithXholandifn&cessary,withasecondrestrictionenzymewhichwillkeepyourgeneofinterestinframewiththeC-terminaltagPleaseNote:Theglu-ala-glu-alasequencemaynotbecompletelyessentialforc

5、leavagebutitdoeshaveaninfluence.IngeneraltheefficiencyofcleavagebythevariouskexinsisaffectedtysequencesbothupstreamanddownstreamoftheL.ys-ArgdibasicmotifbutitseemstovaryfromonemembertoanotherN-terminalsequencesmaynotmatterbutaminoacidsequencesthatconfersecondarystructurethatobscurescleavagesitemayca

6、useaproblem.AnumberofaminoacidsaretoleratedaftertheKEX2cleavagesite:theseincludearomaticaminoacids,smallaminoacids,histidineandmethionine.ProlinewillinhibittheactivityofKEX2.要求目的蛋白中不能含有信號(hào)肽酶切割序列：glu-lys-arg-glu-ala-glu-ala。信號(hào)肽的切除分為兩步：kex2在glu-lys-arg和glu-ala-glu-ala之間初步切除，STE123在兩個(gè)glu-ala之間切割。以pPIC9K

7、為例，pPIC9K是用于在畢赤酵母中的多拷貝整合分泌表達(dá)蛋白的穿梭載體，利用組氨酸缺陷型標(biāo)記進(jìn)行互補(bǔ)篩選。一、信號(hào)肽的2步切割PPIC9K載體帶有自身信號(hào)肽，在分泌過(guò)程中被切除，但是由于切割效率，在目的產(chǎn)物N端帶有35個(gè)左右的信號(hào)肽的氨基酸。設(shè)計(jì)引物時(shí)，為了防止切割不完全，能去掉GluAla兩個(gè)重復(fù)單位，不過(guò)文獻(xiàn)報(bào)道多個(gè)Glu會(huì)提高kex2的切割效率。要求目的蛋白中不能含有信號(hào)肽酶切割序列：glu-lys-arg-glu-ala-glu-ala。信號(hào)肽的切除分為兩步：kex2在glu-lys-arg和glu-ala-glu-ala之間初步切除，STE123在兩個(gè)glu-ala之間切割。二、是否

8、在3端是帶上一些其本來(lái)的序列？1,首先，你要保證表達(dá)后細(xì)胞內(nèi)信號(hào)肽酶能正確識(shí)別原來(lái)的位點(diǎn)，即此位點(diǎn)是不能隨便增減的，否則信號(hào)肽不能正常切割，在這個(gè)基礎(chǔ)上你再?zèng)Q定3端其本來(lái)序列的去留。2，一般5端多出一些氨基酸不會(huì)明顯影響到你的目的蛋白的活性，我們平時(shí)在做酵母表達(dá)時(shí)，信號(hào)肽切割后，目的蛋白往往有幾個(gè)多余的AA,這對(duì)活性好象沒什么影響，雖然如此，但你還得注意多余AA的性質(zhì),不能過(guò)酸或過(guò)堿。三、重組pPIC9K/GS115表達(dá)出目的條帶后，確定信號(hào)肽是否切除完全的方法：1、N端測(cè)序2、先查查你的氨基酸序列里有沒有糖基化位點(diǎn)，如果有就只能N端測(cè)序了。如果沒有，看分子量大小。信號(hào)肽中有kex2和ste1

9、3兩個(gè)酶切位點(diǎn)，現(xiàn)在擔(dān)心的是kex2的位點(diǎn)酶切完全了，但ste13的兩個(gè)酶切位點(diǎn)glu，ala重復(fù)序列沒有切干凈，如果這兩個(gè)氨基酸沒有切除完全的話，分子量差異不大，兩個(gè)氨基酸的差異應(yīng)該看不出來(lái)，一般來(lái)說(shuō)，N端多一兩個(gè)AA應(yīng)該對(duì)蛋白的活性沒有太大影響。western檢測(cè)有兩個(gè)條帶的問(wèn)題，可能是目的蛋白有一部分被降解了。四、附：信號(hào)肽切割原理和優(yōu)化切割ExpressionofYourRecombinantProteinwithaNativeN-terminusIfyouwishtohaveyourproteinexpressedwithanativeN-terminus,youshouldclon

10、eyourgeneflush（直接地）withtheKex2cleavagesite.UsePCRtorebuildthesequeneefromtheXhoIsiteatbp1184-1189totheargininecodonatnucleotides1193-1195.Remembertoincludethefirstaminoacidofyourprotein,ifnecessary,forcorrectfusiontotheKex2cleavagesite.SignalSequenceProcessingTheprocessingofthe-factorsignalsequeneei

11、npPICoccursintwostepsThepreliminarycleavageofthesignalsequencebytheKEX2geneproduct,finalKex2cleavageoccurringbetweenarginineandglutamineinthesequenceLys-Arg*Glu-Ala-Glu-Ala,where*isthesiteofcleavage.TheGlu-AlarepeatsarefurthercleavedbytheSTE13geneproduct.OptimizationofSignalCleavageInSaccharomycesce

12、revisiae,ithasbeennotedthattheGlu-AlarepeatsarenotnecessaryforcleavagebyKex2,butcleavageafterGlu-Lys-ArgmaybemoreefficientwhenfollowedbyGlu-Alarepeats.AnumberofaminoacidsaretoleratedatsiteXinsteadofGluinthesequenceGlu-Lys-Arg-X.Theseaminoacidsincludethearomaticaminoacids,smallaminoacids,andhistidine.Proline,however,willinhibitKex2cleavage.FormoreinformationonKex2cleavage,pleasesee(Brakeetal.,1984).Thereare