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1、ISSN100727626中国生物化学与分子生物学报2004年6月CN1123870PQChineseJournalofBiochemistryandMolecularBiology20(3):376～382垂体腺苷酸环化酶激活肽基因合成表达和产物纯化与鉴定3余榕捷,洪岸,张玲,周天鸿,戴云,高媛(暨南大学生物工程研究所,广州510632)摘要为利用基因工程技术获得垂体腺苷酸环化酶激活肽(pituitaryadenylatecyclaseactivatingpolypeptide,PACAP),根据大肠杆
2、菌的密码偏好性,设计并人工合成编码38个氨基酸的PACAP基+因.克隆到表达载体pET235b(+),构建重组质粒pET2PACAP,转化大肠杆菌BL21(DE3)pLysS.实现纤维素结合域(cellulosebindingdomain,CBD)与PACAP融合蛋白的表达,并在两者之间引入(凝血)因子Ⅹa识别位点(Ile2Glu2Gly2Arg↓).融合蛋白CBD2PACAP经纤维素亲和层析纯化后,因子Ⅹa酶切释放PACAP.在因子Ⅹa识别位点前引入7个氨基酸的柔性短肽(Gly2Thr2Gly2Gly2
3、Gly2Ser2Gly)明显提高了融合蛋白对因子Ⅹa的敏感性.HPLC进一步纯化得到纯度大于95%PACAP多肽.所得的PACAP多肽的Western印迹鉴定为阳性;激光飞行质谱测定分子量结果与理论值相符.生物活性分析表明,所制备的PACAP具有促进胰腺癌细胞株SW1990胞内cAMP合成的活性.关键词垂体腺苷酸环化酶激活肽,因子Ⅹa,基因合成,纯化与鉴定中图分类号Q786GeneSynthesis,ExpressionofPituitaryAdenylateCyclaseActivatingPolype
4、ptideandItsPurificationandIdentification3YURong2jie,HONGAn,ZHANGLing,ZHOUTian2hong,DAIYun,GAOYuan(Bio2engineeringInstituteofJinanUniversity,Guangzhou510632,China)AbstractToproducepituitaryadenylatecyclaseactivatingpolypeptide(PACAP)usinggeneengineeringtec
5、hnology,agenecodingPACAPwasdesignedandsynthesizedaccordingtothepreferenceofE.coli,andclonedintotheexpressionvectorpET235b(+).TherecombinantplasmidpET2PACAPwereconstructedand+transformedintoE.coliBL21(DE3)pLysS.AfusionproteinwitharecognizedsiteoffactorⅩabe
6、tweenCBD(cellulosebindingdomain)andPACAPwasexpressedandpurifiedbycelluloseaffinitychromatography.PACAPwasreleasedbythecleavageoffactorⅩa.Ashortflexiblepeptide(Gly2Thr2Gly2Gly2Gly2Ser2Gly),wasaddedbeforetherecognizedsitebyfactorⅩatoimprovethesensitivityoff
7、usionproteintofactorⅩa.PACAPwithover95%puritywaspurifiedbyHPLCandidentifiedbyWesternblotting.Lasertime2of2flyingmassspectrumshowedthatthemolecularweightwas4536.8asexpected.ThepreliminarybioactivityassayindicatedthattheproducthadtheactivitypromotingcAMPsyn
8、thesisinthecelllineSW1990ofhumanpancreascarcinoma.Keywordspituitaryadenylatecyclaseactivatingpolypeptide(PACAP),factorⅩa,genesynthesis,purificationandidentification垂体腺苷酸环化酶激活肽(pituitaryadenylate收稿日期:2003212208,接受日期:
