1、The Gateway Cloning System,Gene Expression,Contents,Description of destination vectorsExamples of expression clones in destination vectorsEffects of att sites on prokaryotic and mammalian expressionHow to create your own destination vector.,Invitrogen Proprietary & Confidential,2,Choice of the expre
2、ssion system,Easy of use,Cost of media and Equipment,Pos-translational Modifications,(Probability of protein function),Time Requirement,Invitrogen Proprietary & Confidential,3,Native protein expression,N-terminal fusion protein expression,C-terminal fusion protein expression,Cell free,Mammalian,Inse
3、ct,Yeast,Virus,Destination vectors for protein expression,E. coli,Invitrogen Proprietary & Confidential,4,Examples of Destination vectors,For the expression of C-terminal fusion or native proteins,For expression of N-terminal fusion proteins,Invitrogen Proprietary & Confidential,5,Methods for Obtain
4、ing Gateway Expression Clones,Entry Clone,attL2,Gene,attL1,Destination Vector,attR2,ccdB,attR1,+,LR Reaction,+,TOPO Expression,vector,attB2,attB1,PCR Product,Gene,TOPO Reaction,Expression Clone,attB2,Gene,attB1,Invitrogen Proprietary & Confidential,6,* positives/total,Transfer of the CAT Gene into M
5、ultiple Destination Vectors,Invitrogen Proprietary & Confidential,7,Effect of att sites on prokaryotic protein expression,pET300/NT-Dest,pET302 NT-His,MM = MagicMedia LB = Luria-Bertani medium + 1mM IPTG,Invitrogen Proprietary & Confidential,8,Effect of att sites on prokaryotic protein expression,In
6、vitrogen Proprietary & Confidential,9,Effect of att sites on mammalian protein expression,Invitrogen Proprietary & Confidential,10,Effect of att sites on mammalian protein expression,* Extracts normalized for total protein,Invitrogen Proprietary & Confidential,11,Gateway Conversion Kit,Your destinat
7、ion vector,R1,R2,Gateway, Cassette,R1,R2,+,Conversion cassettes can be used with any vector or system, even the proprietary ones,Promoter,Invitrogen Proprietary & Confidential,12,In silico cloning using Vector NTI AdvanceTM 10.3,Primers for PCR reaction,Cloning Strategy,DNA of interest,Invitrogen Proprietary & Confidential,13,PCR,Gene,synthesis,ORF,collection,Library,Your,Source,Gateway Greatly Facilitates Protein Expression Studies,
