巴斯德毕赤酵母表达外源蛋白的优化策略[1]

生物技术通报BIOTECHNOLOGYBULLETIN201012樊英1李天保1杨秀生1王建华2滕达2(1,266002;2,100081):20100624:,,,,;Emai:lfy_fy123@126com:巴斯德毕赤酵母(Pichiapastoris)表达系统已成为外源蛋白最理想的表达系统之一,诸多的优点体现了其广泛的研究价值和应用价值综述了Ppastoris表达外源蛋白时在载体选择与利用外源基因改造翻译后修饰及表达稳定性等方面的优化策略,以加速其应用:巴斯德毕赤酵母外源蛋白优化表达StrategiesforExpressionOptimizationofHeterologousProteinsinPichiapastorisFanYing1LiTianbao1YangXiusheng1WangJianhua2TengDa2(ShandongProvinceMaricultureResearchInstitute,ShandongProvinceKeyLaboratoryforDiseaseControlofMariculture,Qingdao266002;2FeedResearchInstitute,ChineseAcademyofAgriculturalSciences,Beijing100081)Abstrac:tPichiapastorishasbeendevelopedintooneofhighlysuccessfulsystemsforexpressionofvariousheterologousproteins,manyadvantagesofwhichrevealwidevaluesinresearchandapplication.ThearticlesummarizedthosefactorsandtheiroptimizationthatinfluenceexpressionofheterologousproteinsinPichiapastoris,includingthechoiceandutilizationofvectors,modifyingheterologousgenes,posttranslationalmodificationandexpressionstability,thusbeingabletoaccelerateitsapplication.Keywords:PichiapastorisHeterologousproteinsOptimizationexpression,Ppastoris,Ppastoris[1-3]1970,KoichiPpastoris;,PhillipsPetroleumPpastoris,,Ppastoris,FDA,,Ppastoris,[4-6],400Ppastoris,,,(Marsupenaeusjaponicus)SKPI(shrimpKunitztypeproteaseinhibitor)Ppastoris,,SKPI[7]Ppastoris,,Ppastoris,1,SKPIpGEX4T2pETHis,生物技术通报BiotechnologyBulletin201012pPIC9K[7]Ppastoris,pPIC9KpPICZpAO815pPIC6CpGAPZ11启动子的选择Ppastoris1(alcoholoxidase,AOX1),,[8]pPICZA(Acipensersinensis)(cystatin),AOX1cystatin215mg/L(whitespotsyndromeviru,WSSV)P1D3pPIC9KPpastorisGS115,AOX1,302mg/L,[9],AOX1GAP(glyceraldehyde3phosphate),,,Ppastoris[10],pGAPZ,pGAPZBGHGAP50mg/L;,,Delroisse[11]Triboliumcastaneum(carbosylesterase)GAPAOX1Ppastoris,GAP80mg/L,AOX1(40mg/L)AOX2,AOX197%,AOX1AOX2;FLD1(formaldehydedehydrogenase)ICL1(isocitratelyase),[12],[13]12信号肽的选择与改造,,(sCTGly)Ppastoris,10mg/L[15]PHO1(acidphosphatase)Kex2,[16][17][18]Akira[14](Saccharomycescerevisiae)PpastorisPHO1AOX1humanmidkine,281502mg/L,,,,Sagiya[19]NH,10,240mg/L[20]N(NTTEEGEPK)(DDDDK),49[21]pPIC9KfactorNPpastorisAOX1NAIP,Kex2EEAEAEAEPK,8,120mg/L[22]pPIC9KXhoI,,;,,,,Ppastoris,Kex2EA13载体选择标记整合位点和方式,,pPIC9KHIS4kanr,,;pAO815HIS4,42201012:,:,,HIS4SUC2ARG4URA3;,,Kanr(G418)bler(Zeocin),Ppastoris,AOX1,,,aox1,Mut+;,aox1,Muts,221密码子优化,,,,mRNA,pPIC9K,Ppastoris1,31,4102,GS115,7L250mg/L,50[23]22调整GC/AT含量GC/AT,GC/AT,;AT,,AT,,AT30%-55%;GC/ATAkio[24](glucanphosphorylase)AT57%44%,AT,GP100mg/L23优化基因5UTR组成及末端序列mRNA5UTRAOX1mRNA5UTR,5UTRAUG,AUGmRNA,DNAN,N,[25]NTB,BL21GS115TBStreptomycesolivaceoviridisXYNB,6NQ6ET28ETB,AA,G22PS31PN10HN11DTB,TB24基因剂量()[26],[27]eg1,,EG134,88mg/mL;,pAO815,111,19mg/L250mg/L,[28];[29];PCR(overlapextensionPCR),;,[30],,mRNA25基因融合,,,[31],WSSV43生物技术通报BiotechnologyBulletin201012(Growthhormone,GH)WSSVVP28pPIC6c,,3Ppastoris,ON[3]PpastorisP1D3,[32],;PpastorisN,/O,N[33],endoglycisidase,[34]PpastorisN,NN,,PpastorisPpastoris,,Singh[35],N,4Nikolay[36]N,(Equistatin,EI)PpastorispPIC9KMCS(TACGTAGAATTC),EI4-10,166g/L,,MCSPpastoris4:(1),SMD1168(his4pep4),A,[37]GS115KM71SMD11681,6,22330341235U/mL,GS115,(2)(PMSFPepstatin)Woo[38]1%1-3mmol/LPMSF,G4S35mg/L,,(3)[39]PpastorispPIC9KArresten,6ACCCCA,(4)Ppastoris(pH),,[40][41](pH)2(MvhIL2),3,30%,,25,Ppastoris,,,,,,,,[1],,,.PichiapastorisGS115.,2009(12):19621968.44201012:[2],,,.LL37SMD1168.,2010(2):98101.[3],,,.P1D3.,2006,22(6):973978.[4]ZhangWK,FanQL,SongLH.TheadvancesandapplicationintheexpressionofheterologousgeneinPichiapastoris.ChinaBiotechnology,2006,26(1):8791.[5]..:,2002,30(4):6163.[6]..:,2009(12):3739.[7],,.Kunitz.,2008,24(3):500503.[8]MaDM,BaiJJ,JianQ,eta.lExpressionofchinesesturgeoncystatininyeastPichapastorisanditsproteinaseinhibitoryactivityanalysis.ChineseJournalofBiotechnology,2003,19(5):598602.[9]YangY,ZhangM,YuanL,eta.lExpressionofsinglechainfragmentvariableP1D3antibodyagainstshrimpwhitespotsyndromevirusinPichiapastoris.ChineseJournalofBiotechnology,2006,22(6):973978.[10],,,..,2003,30(4):301306.[11]DelroisseJM,MarieD,GilsoulJJ,eta.lExpressionofasyntheticgeneencodingaTriboliumcastaneumcarbosylesteraseinPichapastoris.ProteinExpression&Purification,2005,42:286294.[12]SueMP,MarianaLF,BrianM,eta.lHeterologousproteinproductionusingthePichiapastorisexpressionsystem.Yeast,2005,22:249270.[13],,,.AOX1GAPSAMS.,2008,38(4):2430.[14]AkiraM,YumikoTA.ComparisonofthreesignalsforsecretoryexpressionofrecombinanthumanmidkineinPichiapastoris.BiosciBiotechnolBiochem,2001,65(10):22912293.[15],,..,2007,38(6):411415.[16]CaleraJA,Paris,MonodS,eta.lCloninganddisruptionoftheantigeniccatalasegeneofAspergillusfumigatus.InfectionandImmunity,1997,65:47184724.[17]ChungBH,NamSW,KimBM,eta.lCommunicationtotheeditorhighlyefficientsecretionofheterologousproteinsfromSaccharomycescerevisiaeusinginulinasesignalpeptides.BiotechnologyandBioengineering,1996,49:473479.[18]ZhangZQ,LiJP,HuY.Secretionofrecombinantproteinsinmammaliancellsdirectedbygrowthhormonesignalpeptide.ChinJBiochemandMolecularBio,l2005,21(2):282286.[19]SagiyaY,YamagataH,UdakaS.Directhighlevelsecretionintotheculturemediumoftunagrowthhor