中国药科大学：The construction, fermentation, expression , preparation and analysis of Recombinant L-asparaginase

中國药科大学生命科学与技术学院SCHOOL OF LIFE SCIENCE AND TECHNOLOGYThe construction, fermentation,expression, preparation and analysis ofRecombinant L-asparaginase

The construction, fermentation, expression , preparation and analysis of Recombinant L-asparaginase

U1Construction of recombinant engineeredbacteria.2Purification and analysis of recombinantprotein

1 2 Construction of recombinant engineered bacteria. Purification and analysis of recombinant protein

重组表达门冬酰胺酶川工程菌的构建重组门冬酰胺酶的诱导表达及分离和纯化

2 重组表达门冬酰胺酶II工程菌的构建 重组门冬酰胺酶II的诱导表达及分离 和纯化

从pET28a-AnsB中扩增AnsB基因提取pET22b质粒（多克隆位点含有（带有HindlLI和Ncol酶切位点）HindilI和NcoL酶切位点）凝胶电泳检测分别用HindIII和Ncol双酶切（37C，3h）PCR纯化试剂盒分别纯化酶切产物电泳检测纯化产物浓度1纯化产物用T4连接酶连接（16℃C，保制备大肠杆菌B121感受态温过夜）热激转化连接液（42C.90S）1涂板、筛选重组子（Amp100）模块一重组工提质粒酶切验证程菌的构建酶活力测定

接重组工程菌（pET28a-AnsB）发酵培养，诱导表达1低温离心收集菌体（6000g，4C，15min）用醋酸钠缓冲液悬浮菌体超声破碎收集上清酶溶液155%饱和度硫酸铵沉淀，离心去除杂蛋白留90%饱和度硫酸铵沉淀，离心收集目标蛋白样样装柱（DE52阴离子交换透析除盐离子柱），平衡上柱，用蛋白纯化仪收集蛋白1分析酶活力、蛋白含量、SDS-PAGE电泳分析模块二重组蛋绘制酶活力曲线、蛋白分离曲线白的纯化和分析

PCRExtractionplasmidpET22bpET22b-AnsBAmplifyAnsBgene食Agrosegelelectrophoresis(AGE)testUsingHindIII and NcoI restriction endonucleasedigestplasmid and PCRproductsPurifybothPCRandplasmidpET22bdigestedproductsCultivateE.coliBL21by PCR extraction Kit 几PurifiedproductslinkedbyT4PreparationofcompetentcellbyDNAligase(16°Covernight)using CaCI2methodE.coliBI21transfomationChapter I几ScreentherecombinantbacteriaonLB solid medium(Amp100)1Inoculate single colony in the LB liquid mediumExtractiontheplasmidandconfirmwithAGE

Chapter I pET22b-AnsB PCR Amplify AnsB gene Extraction plasmid pET22b Agrose gel electrophoresis (AGE) test Using Hind III and Nco I restriction endonuclease digest plasmid and PCR products Purify both PCR and plasmid pET22b digested products by PCR extraction Kit Purified products linked by T4- DNA ligase(16℃ overnight) Cultivate E. coli BL21 Preparation of competent cell by using CaCl2 method E. coli Bl21 transfomation Screen the recombinant bacteria on LB solid medium (Amp100) Inoculate single colony in the LB liquid medium Extraction the plasmid and confirm with AGE

InoculaterecombinantbacteriawithpET22b-AnsBintheLBliquidmediumUsingIPTGtofinishinducibleexpressionCentrifugation and collectionofrecombinantbacteria(6000g,4°C,15min)Solidacetatebuffer suspended and usingosmotic shocktoreleasetargetproteinAmmonium sulfatefractionation:90% saturation, collect designed proteinDialysisChapter IISeparatetargetproteinwithNicolumnTest enzyme activity and contentof protein: SDS-PAGE electrophoretic analysisAnalysis drawing enzyme activity curve,protein seperation curve

Inoculate recombinant bacteria with pET 22b-AnsB in the LB liquid medium Using IPTG to finish inducible expression Centrifugation and collection of recombinant bacteria (6000g, 4℃, 15min) Solid acetate buffer suspended and using osmotic shock to release target protein Ammonium sulfate fractionation: 90% saturation, collect designed protein Dialysis Separate target protein with Ni column Test enzyme activity and content of protein: SDS-PAGE electrophoretic analysis Analysis drawing enzyme activity curve, protein seperation curve Chapter II

Ava l(158)Xho l(158)Not l(166)Eagl(166)Hind ill173),Bpu1102 I(80)Sal l(179)Saci(190)EcoRI(192)BamH(198)NcO(220)Msc I(225)BseR(280)Dra ll(5251)BspM I(208)Nde l(288)Xba l(328)Bgl I(302) oigin(5027-5482)SgrA l(433)Sph i(580)PfIM (696)ApaB l(7ee)Sca l(4588)Ap(4038-489Pvu l(4478)Mu I(1114)Bcll(1128)Pst (4353)lacl（764-1843)BstEIl(1295)Bmgl(1323)Bsal(4160)Apal(1325)m1105 (4108)pET-22b(+)(5493bp)BssHIl(1525)Plasmid profile ofHpa I(1620)pET22bAwNl(3631)OPshA l(1059)Psp5Il(2221)BspLU11(3215)Bpu10l(2321)Sapl(300g)Bst1107((29e6)BspGI(2741)Tth111 (29e0)

Plasmid profile of pET22b

UpET-22b(+)cloning/expression regionT7promoterprimer#00348-317promoterBgilllacoperatorXbalrbsAGATCTCCATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGABspMIMsclNcolpelB leaderNdelBamHIEcoRISacTATACATATCAAATACCTGCTGCCGACCCCTGCTGCTCGTCTCCTGCTCCTCGCTGCCCACCCGCCGATGCCCATCGATATCGGAATTAATTCGGATCCGAATTCGAGCTCCMetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnProAlaMetAlqMetAsplleGlyl leAsnSerAspProAsnSerSerSerEag!signalpeptidaseAvarHis-TagBpu/11021SallNotlXhoiHind IllGTCGACAAGCTTGCGCCCGCACTCGAGCACCACCACCACCAC CACTGAGATCCGGC.TGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC TGCCACCGCTGAGCAATAACValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd17terminatorprimer#ee337-3T7terminatorTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG

pET-22b(+)cloning/expression region

#Chapter 1The Construction of Recombinant AsparaginaseII Engineering Bacteria

Chapter 1 The Construction of Recombinant Asparaginase II Engineering Bacteria