1. The Gateway® Cloning System Gene Expression 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. Invitrogen Proprietary & Confidential 2 Choice of the expression system Cell-free BacteriaYeastInsectMammalian Easy of use Cost of media and Equipment Pos-translational Modifications (Probability of protein function) Time Requirement

3. Invitrogen Proprietary & Confidential 3 Native protein expression N-terminal fusion protein expression C-terminal fusion protein expression Cell free Mammalian Insect Yeast Virus Destination vectors for protein expression E. coli

4. 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

5. Invitrogen Proprietary & Confidential 5 Methods for Obtaining Gateway® Expression Clones Entry Clone attL2GeneattL1 Destination Vector attR2 ccdB attR1 + LR Reaction + TOPO ® Expression vector attB2attB1 PCR Product Gene TOPO ® Reaction Expression Clone attB2GeneattB1

6. Invitrogen Proprietary & Confidential 6 Destination Vector/ApplicationColoniesBackgroundAnalysis* E. coli Native15,00004/4 6xHis-fusion10,65004/4 GST-fusion9,20004/4 Thioredoxin-fusion11,00004/4 Sequencing + Strand13,95004/4 Sequencing - Strand8,95004/4 Insect Native protein7,800154/4 6xHis 6,350304/4 Mammalian CMV-promoter7,95004/4 SFV4,50004/4 Tet-regulated promoter6,35004/4 * positives/total Transfer of the CAT Gene into Multiple Destination Vectors

7. 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

8. Invitrogen Proprietary & Confidential 8 Effect of att sites on prokaryotic protein expression

9. Invitrogen Proprietary & Confidential 9 Effect of att sites on mammalian protein expression Extracts normalized for total protein Western blot probed with anti V5 antibody 1 2 3 LacZ lacZ CMVp V5His 1) pCDNA3.1/lacZ/V5His 2) pDEST40/lacZ/V5His CMVp lacZ attB1 V5His 3) MultiSite CMV/lacZ/V5His CMVp lacZ V5His attB2 attB3attB1attB4

10. Invitrogen Proprietary & Confidential 10 Effect of att sites on mammalian protein expression * Extracts normalized for total protein Gateway (G) MultiSite CMV/ORFV5His/ CMVp ORF V5His attB2attB3attB1attB4 Non Gateway (NG) pCDNA3.1 D-TOPO/ORF ORF CMVp V5His

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

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

13. Invitrogen Proprietary & Confidential 13 PCR Gene synthesis ORF collection Library Your Source Gateway® Greatly Facilitates Protein Expression Studies