毕赤酵母发酵生产重组人血清白蛋白高密度培养条件的研究

3042003JOURNALOFBEIJINGUNIVERSITYOFCHEMICALTECHNOLOGYVol.30,No.420031212213(1.,100029;2.,100080):(NH4)2SO4PTM1pH,PTM1,6mL/L3g/L,3g/L,pH61061510mL/L0105%:;;:Q939.9:2002209222:,1977,3E2mail:[e-mail],60%,(,,,),,,,,[1],,,(HSA),HSA,,,HSA[2],,,,HSA,[3],,1111PichiapastorisGS115,aox+his+,Paox1,pPIC9K,HSA,()11211211YPD(yeastpowder1%,tryptone2%,2%)2%1121220g/L,(NH4)2SO45144g/L,MgSO47H2O14176g/L,K2SO413192g/L,CaSO401272g/L,011mol/L(PBSpH=6)11213PTM1[4]11330,18h,10%,28h,30,180r/min,50mL/500mL114:600nmOD600=OD;:2[5];(OD595):[6];:;(rHSA):SDS2PAGE[7]2211(NH4)2SO4,(NH4)2SO4,1(NH4)2SO4(NH4)2SO450mg/L,,(NH4)2SO4310g/LPichiapastoris,pHpH,,124h(NH4)2SO4,2g/L,w1(NH4)2SO4rHSAFig.1Effectof(NH4)2SO4concentrationonbiomassandtheexpressionofrHSA212pH,pH,pH310,410,510,610,615,pH,,22,rHSApH610,rHSA,KOHpH,610615,rHSA2pHrHSAFig.2EffectofpHontheexpressionofrHSA213PTM121311PTM1PTM1PTM1,,PTM1rHSA,3,11910mg/L19213mg/L,6116%Ca2+,Fe3+,Zn2+,Mg2+,Co2+I-Mo2+,,;24h,,60h,,3PTM1Fig.3EffectofPTM1ontheexpressionofrHSA21312PTM1rHSA,PTM118h,,PTM1,,,,PTM1,12hPTM1,PTM1011%,012%,016%,112%6220034,12hPTM1V012%016%,4PTM1Fig.4EffectofPTM1ontheexpressionofrHSAduringtheperiodofinductment214,,,55Fig.5EffectofoleicacidontheexpressionofrHSA5,0105%,,,,0105%215,AOX1(),rHSA[8]Pichiapastoris,,,,rHSA,66Fig.6EffectofmethanolontheexpressionofrHSA6,10mL/L,5mL/L40%,,11,1%,115%215%,,1Table1Effectofmethanolconcentration/%/%(OD600)/(mg/L)w()/(mg/L)015052100160611006113018931150109985715018092100122675610016882150146934719016291,2,3,5,70105%,1%,115%,2%,215%;411g/L;61Marker7rHSAFig.7SDS2PAGEelectrophoresisresultoftheexpressedproteins7SDS2PAGE,724:,75%3,,,,[1],,,.Pichiapastoris[J].,2000,32(1):59-62[2],,,.[J].,2000,20:39-42[3]CreggJM,VedvickTS,RaschkeWC.Recentad2vancesintheexpressionofforeigngenesinPichiapas2toris[J].Bio/Technology,1993,11(8):905-910[4]ClareJJ,RaymentFB,BallantineSP,etal.High2levelexpressionoftetanustoxinfragmentCinPichiapastorisstrainscontainingmultipletandemintegrationsofthegene[J].Bio/Technology,1991,9(4):455-460[5].[M].:,1983[6]J,EF,J.[M]..,.:,1998[7],,,.[M].:,1992[8]CreggJM,MaddenKR,BarringerKJ,etal.Func2tioncharacterizationofthetwoalcoholoxidasegenefromtheyeastPichiapastoris[J].MolCellBiol,1989(9):1316-1323HighdensitycultureconditionsofrecombinanthumanserumalbumfromPichiapastorisLIUYan2li1LIUYong2dong2WANGYan2hui1WANGYun2shan2SUZhi2guo2MARun2yu1(1.TheKeyLaboratoryofScienceandTechnologyofControllableChemicalReactions,MinistryofEducation,BeijingUniversityofChemicalTechnology,Beijing100029;2.TheStateKeyLabofBiochemicalEngineering,theInstituteofProcessEngineeringoftheChineseAcademyofSciences,Beijing100080,China)Abstract:ThehighdensitycultivationofacombinedstrainofproducingrHSA(recombinanthumanserumalbu2min)fromPichiapastoriswasstudied.Withthesameofthesynthesizedmedium,theinfluencesof(NH4)2SO4,PTM1,oleicacid,methanolandpHontheexpressionofrHSAwereinvestigated.ItwasdiscoveredthatPTM1significantlyimprovedtheexpressionofrHSAandtheoptimumconcentrationofPTM1was6mL/L.ThehighestproductivityofrHSAwasobtainedwhentheconcentrationof(NH4)2SO4was3g/Landtheproteinexpressionwasdepressedwhentheconcentrationwashigherthan3g/Linthecourseofinducement.Theopti2mumconcentrationofmethanolwas10mL/L.TheexpressionofrHSAwasdepressedwhenpHwaslowerthan610andtheoptimumconditionwaspH610615.Oleicacidimprovedtheproteinexpressiongreatlyandtheoptimumconcentrationwas0105%.Theabovedatacouldbeusedtodirectthefed2batchfermentation.Keywords:Pichiapastoris;recombinanthumanserumalbumin;highdensitycultivation()822003