信號肽酶切割

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肽酶切割序列：glu-lys-arg-glu-ala-glu-ala。信號肽的切除分為兩步：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蛋白的穿梭載體，利用組氨酸缺陷型標記進行互補篩選。一、信號肽的2步切割PPIC9K載體帶有自身信號肽，在分泌過程中被切除，但是由于切割效率，在目的產(chǎn)物N端帶有35個左右的信號肽的氨基酸。設(shè)計引物時，為了防止切割不完全，能去掉GluAla兩個重復單位，不過文獻報道多個Glu會提高kex2的切割效率。要求目的蛋白中不能含有信號肽酶切割序列：glu-lys-arg-glu-ala-glu-ala。信號肽的切除分為兩步：kex2在glu-lys-arg和glu-ala-glu-ala之間初步切除，STE123在兩個glu-ala之間切割。二、是否

8、在3端是帶上一些其本來的序列？1,首先，你要保證表達后細胞內(nèi)信號肽酶能正確識別原來的位點，即此位點是不能隨便增減的，否則信號肽不能正常切割，在這個基礎(chǔ)上你再決定3端其本來序列的去留。2，一般5端多出一些氨基酸不會明顯影響到你的目的蛋白的活性，我們平時在做酵母表達時，信號肽切割后，目的蛋白往往有幾個多余的AA,這對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),不能過酸或過堿。三、重組pPIC9K/GS115表達出目的條帶后，確定信號肽是否切除完全的方法：1、N端測序2、先查查你的氨基酸序列里有沒有糖基化位點，如果有就只能N端測序了。如果沒有，看分子量大小。信號肽中有kex2和ste1

9、3兩個酶切位點，現(xiàn)在擔心的是kex2的位點酶切完全了，但ste13的兩個酶切位點glu，ala重復序列沒有切干凈，如果這兩個氨基酸沒有切除完全的話，分子量差異不大，兩個氨基酸的差異應(yīng)該看不出來，一般來說，N端多一兩個AA應(yīng)該對蛋白的活性沒有太大影響。western檢測有兩個條帶的問題，可能是目的蛋白有一部分被降解了。四、附：信號肽切割原理和優(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