信号肽序列对毕赤酵母表达外源蛋白质的影响

ISSN0582-9879ACTABIOCHIMICAetBIOPHYSICASINICA2003,35(2):154-160CN31-1300/Q:2002-08-21　　:2002-10-16(No.993913002)＊:Tel,021-62205704;Fax,021-62209988;e-mail,[e-mail]熊爱生　彭日荷　李　贤　范惠琴　姚泉洪＊　郭美锦1　张嗣良1(、,201106;1,200237)　　、,,。,。αMF4I,MF4IN1～10Aox1N,10,10。,NA、I、P;α,5,90mg/L。MF4IEEAEAEAEPK10,,35%,120mg/L,pPCI9K8。　　;;;　　(Pichiapastoris),。、,。,,、、、;、、。　　,,,[1,2];[3,4]。。　　、、,。mRNA5′(5′-UTR)。5′mRNA。mRNA5′-UTRAox1mRNA5′-UTR。(HAS)Aox1mRNA5′-UTR,HSA50[5]。α(MFα)[6～9],MFαEEAEAEAEPK102[10]。[11]。Aox1Aox1,ATGAox1ATG。　　。PCR[12,13],。1　(MaterialsandMethods)1.1　1.1.1　菌株与质粒　　[Pichiapas-toris(His-Mut+)](ATCC)。E.coliDH5α、pBluescriptSKPromega。pPIC9KInvitrogen。1.1.2　试剂耗材　　PCR,、T4PyrobestDNATaKaRa。DNAPhar-macia。G418、EndoH、(、、)Sigma,AR。1.2　1.2.1　信号肽序列的化学合成　　GenBank(Z46233)[14]Aox1[15],[16],。,5′,3′。20～25bp(1)。PCR[12,13](1)。1.2.2　DNA操作　　[17]。DNAABI377DNA。。1.2.3　酵母各表达载体构建　　,HindIIIXhoI,[18],。1.2.4　酵母的电击转化及筛选　　500mLYPD(20g/L、10g/L、20g/L)30℃18h,A1.7,5000r/min,500mL、250mL,,20mL1mol/L。0.5mL,。,BglII,2μg50μL,5min,Bio-RedGenePulser,2.5kV,25μF。,1.0mL1.0mol/L,200μL[186g/L、20g/L、13.4g/L(YNB)、0.05g/L、0.05g/L、0.05g/L、0.05g/L、0.05g/L、20g/L],30℃。MM(13.4g/LYNB、0.4mg/L、5g/L、15g/L)MD(13.4g/LYNB、0.4mg/L、20g/L、15g/L),30℃2,MDMM。MDG4180.5μg/LMD。G418MD,MD[19]。1.2.5　重组酵母的培养及诱导表达　　20mLBMGY(10g/L、20g/L、13.4g/LYNB、0.4mg/L、10g/L),30℃,,20mLBMMY(0.5%BMGY),30℃36h。1.2.6　高表达菌株筛选及SDS-PAGE电泳分析　　,30℃A3,0.5%36h。5μLSDS-PAGE。,30℃A4～5,36h。10μLSDS-PAGE,。,[20],。100μg3000uEndoH,37℃5h,SDS-PAGE[21]。　　。[22]。(u):37℃,1nmol/L1u。2　(Results)2.1　　　1PCR,1,PyrobestDNA15,:XHT1、XHT2、XHT3、XHT4、XHT5、XHT6、XHT7、XHT8、XHT9、XHT10、XHT11、XHT12、XHT13、XHT14XHT15(2)。XHT1ATGBamHI(GGATCC),,(notuse-biascodon),。XHT2～15155Feb.,2003:Fig.1　ThenucleotidesequenceofsignalpeptidesynthesisbysuccessivePCREachofchemicallysyntheticoligonucleotideswasabout90bp.Alltheseprimerscanassembledcorrectlybyone-stepPCRwhen1-2nginnerprimersand100-200ngexternalprimerswereblendedtogether.ThosePCRexternalprimerscontainsuitablerestrictioncleavagesitesforcloning.(usebiascodon)。XHT3BamHI,XHT4BamHI。XHT4～13BamHI,MF4IATGAox1ATG1～10,A、I、P、E、E、F、D、I、LV。,,AIP。MF4IEEAEAEAEPK10,,XHT14。,,XHT15,:Fig.2　ComparisonofnucleotidesequencesbetweensyntheticsignalpeptidesequenceandthesequencefrompPCI9KTheunderlinedregionaredeletedthesiteofBamHI.Theboxedregionen-codesA、I、P、E、E、F、D、I、LandVaminoacidresidues.Thelower-caseregioninboldencodesEEAEAEAEPKaminoacidresidues.、,AIPEEAEAEAEPK,。pBlue-scriptSK,DNA,,(2)。2.2　Table1　Primersforenzymaticsynthesisofsignalpeptidesequence　　TheunderlinedregionsareHindIIIandXhoIsites.TheboxedregionencodesA、I、P、E、E、F、D、I、LandVaminoacidresidues.TheshadowedregionencodesEEAEAEAEPKaminoacidresidues.156　　　ACTABIOCHIMICAetBIOPHYSICASINICAVol.35,No.2　　　　　Table2　ThenameandcharacterizationofthefifteenconstructionofplasmidsNamePrimersCharacterizationofthesignalHindIIIATGXhoIacidphygene　pPCI9K　XHT1S11、S21AAAAACG(MRF)Notusebiascodon　XHT2S11、S3、S5、S6、S4、S2AAGGATCCAAACG(MRF)Usebiascodon　XHT3S1、S3、S5、S6、S4、S2AAAAACG(MRF)Usebiascodon　XHT4S1、S31、S5、S6、S4、S2AAAAACG(MARF)Usebiascodon　XHT5S1、S32、S5、S6、S4、S2AAAAACG(MAIRF)Usebiascodon　XHT6S1、S33、S5、S6、S4、S2AAAAACG(MAIPRF)Usebiascodon　XHT7S1、S34、S5、S6、S4、S2AAAAACG(MAIPERF)Usebiascodon　XHT8S1、S35、S5、S6、S4、S2AAAAACG(MAIPEERF)Usebiascodon　XHT9S1、S36、S5、S6、S4、S2AAAAACG(MAIPEEFRF)Usebiascodon　XHT10S1、S37、S5、S6、S4、S2AAAAACG(MAIPEEFDRF)Usebiascodon　XHT11S1、S38、S5、S6、S4、S2AAAAACG(MAIPEEFDIRF)Usebiascodon　XHT12S1、S39、S5、S6、S4、S2AAAAACG(MAIPEEFDILRF)Usebiascodon　XHT13S1、S310、S5、S6、S4、S2AAAAACG(MAIPEEFDILVRF)Usebiascodon　XHT14S1、S33、S5、S6、S41、S2AAAAACG(MRFEEAEAEAEPK)Usebiascodon　XHT15S1、S33、S5、S6、S41、S2AAAAACG(MAIPRFEEAEAEAEPK)Usebiascodon　　XhoINotIpPCI9K,。15HindIIIXhoIpYPX98,,15(3)。。2.3　　　BglII16。His(His-),His,Fig.3　Constructionofplasmidsofinclusionelevenkindsofsignalpeptide。,His+,200。,,,。MMMD,,30。16MDG4180.5μg/LMD。G418MD,MD,10BMGY,30℃A3.0,,BMMY36h,SDS-PAGE。pPCI9KXHT1～155%,,。,。2.4　　　16,EndoH,SDS-PAGE,。64kD,(4)。,pPCI9K15mg/L,2.2u/mL。XHT1、XHT2、XHT3、XHT4、XHT5、157Feb.,2003:Fig.4　SDS-PAGEanalysisofphytaseexpressioninPichiapastorisM,proteinmolecularweightstandards(kD);0,purifiedphytase;1,ex-pressionvectorpPCI9K;2-16,expressionvectorsXHT1-15.XHT6、XHT7、XHT8、XHT9、XHT10、XHT11、XHT12、XHT13、XHT14XHT15pPCI9K。Aox1,93%;,106%;ATGAox1ATG1～10(A、I、P、E、E、F、D、I、LV),13,35。MF4IEEAEAEAEPK10,2.4。XHT15,,120mg/L,17.3u/mL,pPCI9K8.007.85。(3)。3　(Discussion)　　。10,。,。:;;mRNA5′3′(5′-UTR3′-UTR);ATG;Table3　ThecomparisonoftheexpressionvalueamongthoseplasmidsLaneExpressionlevel(mg/L)Relativity(%)Phytaseactivity(u/mL)Samplename1151002.2pPCI9K2291934.5XHT13312064.7XHT24281864.3XHT357046512.0XHT46402656.4XHT579060013.5XHT68604009.5XHT796543310.5XHT810583859.2XHT911553659.0XHT10126241310.0XHT1113352335.2XHT1214382535.5XHT1315513407.8XHT141612080017.3XHT150purifiedphytase(Sigma)MmarkerAT;;;;、、、。。　　PCR,。5′-UTRMF4IATG,mRNA,mRNA。ATGAox1ATG10,MF4IEEAEAEAEPK10,。,,[5],10,[23],,[21]。G418,。　　,,。PCR,,,,4.8g/L。,、158　　　ACTABIOCHIMICAetBIOPHYSICASINICAVol.35,No.2　　　　　、cryIA(c)Bt,,6～10()。References1　ClareJJ,RaymentFB,BallantineSP,SreekrishnaK,RomanosMA.High-leve