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The Gateway® Cloning System

Gene Expression. The Gateway® Cloning System. Contents. Description of destination vectors Examples of expression clones in destination vectors Effects of att sites on prokaryotic and mammalian expression How to create your own destination vector. Bacteria. Yeast. Insect. Mammalian.

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The Gateway® Cloning System

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  1. Gene Expression The Gateway® Cloning System Contents Description of destination vectors Examples of expression clones in destination vectors Effects of att sites on prokaryotic and mammalian expression How to create your own destination vector.

  2. Bacteria Yeast Insect Mammalian Cell-free Choice of the expression system Easy of use Cost of media and Equipment Pos-translational Modifications (Probability of protein function) Time Requirement Invitrogen Proprietary & Confidential

  3. Destination vectors for protein expression Native protein expression E. coli Cell free Mammalian N-terminal fusion protein expression Insect Yeast Virus C-terminal fusion protein expression 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 Obtaining Gateway® Expression Clones attR1 attR2 attL1 Gene attL2 ccdB + Destination Vector Entry Clone LR Reaction attB1 Gene attB2 Expression Clone TOPO® Reaction attB1 attB2 Gene + TOPO® Expression PCR Product vector Invitrogen Proprietary & Confidential

  6. Transfer of the CAT Gene into Multiple Destination Vectors * positives/total 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 Invitrogen Proprietary & Confidential

  9. 1 3 2 1) pCDNA3.1/lacZ/V5His CMVp lacZ V5His LacZ 2) pDEST40/lacZ/V5His CMVp attB1 attB2 lacZ V5His 3) MultiSite CMV/lacZ/V5His CMVp attB4 attB1 attB2 attB3 lacZ V5His Extracts normalized for total protein Western blot probed with anti V5 antibody Effect of att sites on mammalian protein expression Invitrogen Proprietary & Confidential

  10. Non Gateway (NG) pCDNA3.1 D-TOPO/ORF CMVp ORF V5His Gateway (G) MultiSite CMV/ORFV5His / attB4 attB1 attB2 attB3 CMVp ORF V5His Effect of att sites on mammalian protein expression * Extracts normalized for total protein Invitrogen Proprietary & Confidential

  11. Your linearized vector Gateway® Conversion Kit R2 R1 Gateway ® Cassette R2 R1 + Your destination vector Promoter Conversion cassettes can be used with any vector or system, even the proprietary ones Invitrogen Proprietary & Confidential

  12. In silico cloning using Vector NTI AdvanceTM 10.3 DNA of interest Primers for PCR reaction Cloning Strategy Invitrogen Proprietary & Confidential

  13. Gene Gene Gene Protein Your Application Localization Gene Entry Clone Gene Gene Protein RNAi Purification Gene Gene Protein Cell-Free interaction Gene1 Gene2 Gene3 Gene4 Your Application Gateway® Greatly Facilitates Protein Expression Studies Your Source PCR Library ORF Gene collection synthesis Invitrogen Proprietary & Confidential

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