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1、2005年32(4)微生物学通报25*海栖热袍菌胞外-淀粉酶在Ecoli中的高效表达111,2**王希菊蒋宇邵蔚蓝(江南大学工业生物技术教育部重点实验室无锡214036)1(南京师范大学生命科学学院南京210097)2摘要:为解决密码子偏好性差异造成的表达水平低下问题,采取了3种手段提高海栖热袍菌胞外-淀粉酶在Ecoli中的表达水平。用PCR方法从海栖热袍菌(Thermotogamaritima)基因组DNA中扩增出胞外-淀粉酶A的完整基因amyA,插入表达载体pET-20(b)中构建成质粒pET-amyA;运用基因工程手段将amy
2、A基因富含稀有密码子的信号肽进行切除,将不含信号肽的amyAⅠ基因插入pET-20(b)中构建成质粒pET-amyAⅠ;用PCR法从大肠杆菌基因组中扩增出argU基因,插入pET-amyAⅠ中构建成质粒pET-amyAⅡ。将重组质粒分别转化到EcoliJM109(DE3),并将重组质粒pET-amyAⅠ转化EcoliBL21-CodonPlus(DE3)-RIL。通过IPTG诱导测酶活性:在EcoliJM109(DE3)中表达的重组酶(pET-amyA)、(pET-amyAⅠ)、(pET-amyAⅡ)的酶活分别是16580U/mL、67217U/mL、89045U/mL,在
3、EcoliBL21-CodonPlus(DE3)-RIL中表达的重组酶(pET-amyAⅠ)的酶活是138677U/mL。表明通过这些手段能大幅提高Tmaritima胞外-淀粉酶在Ecoli中的表达水平。关键词:海栖热袍菌,-淀粉酶,argU基因,高效表达中图分类号:Q93文献标识码:A文章编号:0253-2654(2005)04-0025-06Hgh-levelExpressionofExtracellular-Amylaseof*ThermotogaMaritimainEcoli111,2**WANGX-iJuJIANGYuSHAOWe-iLan(The
4、KeyLaboratoryofIndustrialBiotechnologyofMinistryofEducation,SouthernYangtzeUniversity,1Wuxi214036)2(DepartmentofLifeScience,NanjingNormalUniversit,Nanjing210097)Abstract:Thecompletegeneencodingextracellular-amylasewasamplifiedfromthegenomicDNAofThermoto-gamaritimabypolymerasechainreactionThere
5、combinantplasmidpET-amyAwasconstructedbyinsertingtheamplifiedsegmentintotheexpressionvectorpET-20(b)ThesignalpeptideofamyAgeneboundofunoptimalcodonwascutofftoformamyAⅠTherecombinantplasmidpET-amyAⅠwasobtainedbyinsertingtheamyAⅠintothevectorpET-20(b)ThecompleteargUgenewasamplifiedfromthegenome
6、ofEcolibypolymerasechainreactionandwasinsertedintotheplasmidpET-amyAⅠtoformpET-amyAⅡTherecombinantplasmidspET-amyA,pET-amyAⅠ,pET-amyAⅡweretransformedintotheEcoliJM109(DE3)respectivelyandTherecombinantplasmidspET-amyAⅠwastransformedintotheEcoliBL21-CodonPlus(DE3)-RILTherecomb-inantstrainsEc
7、oliJM109(DE3)harbouringpET-amyA,pET-amyAⅠ,pET-amyAⅡandEcoliBL21-Codon-Plus(DE3)-RILharbouringpET-amyAⅠproducedamylaseactivitiesof16580U/mL,67217U/mL,89045U/mL,13877U/mLthroughIPTGinduction,respectivelyKeywords:Ther
