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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.
Invitrogen Proprietary & Confidential2
Choice of the expression system
Cell-free Bacteria Yeast Insect Mammalian
Easy of use
Cost of media and Equipment
Pos-translational Modifications(Probability of protein function)
Time Requirement
Invitrogen Proprietary & Confidential3
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
Invitrogen Proprietary & Confidential4
Examples of Destination vectors
For the expression of C-terminalfusion or native proteins
For expression of N-terminalfusion proteins
Invitrogen Proprietary & Confidential5
Methods for Obtaining Gateway® Expression Clones
Entry Clone
attL2GeneattL1
Destination Vector
attR2ccdBattR1
+LR Reaction
+TOPO® Expression
vector
attB2attB1
PCR Product
Gene
TOPO® Reaction
Expression Clone
attB2GeneattB1
Invitrogen Proprietary & Confidential6
Destination Vector/Application ColoniesBackgroun
dAnalysis
*
E. coli
Native 15,000 0 4/4
6xHis-fusion 10,650 0 4/4
GST-fusion 9,200 0 4/4
Thioredoxin-fusion 11,000 0 4/4
Sequencing + Strand 13,950 0 4/4
Sequencing - Strand 8,950 0 4/4
Insect
Native protein 7,800 15 4/4
6xHis 6,350 30 4/4
Mammalian
CMV-promoter 7,950 0 4/4
SFV 4,500 0 4/4
Tet-regulated promoter 6,350 0 4/4* positives/total
Transfer of the CAT Gene into Multiple Destination Vectors
Invitrogen Proprietary & Confidential7
Effect of att sites on prokaryotic protein expression
pET300/NT-Dest pET302 NT-His
MM = MagicMedia™LB = Luria-Bertani medium + 1mM IPTG
Invitrogen Proprietary & Confidential8
Effect of att sites on prokaryotic protein expression
Invitrogen Proprietary & Confidential9
Effect of att sites on mammalian protein expression
Extracts normalized for total proteinWestern blot probed with anti V5 antibody
1 2 3
LacZ
lacZCMVp V5His
1) pCDNA3.1/lacZ/V5His
2) pDEST40/lacZ/V5His
CMVp lacZattB1 V5His
3) MultiSite CMV/lacZ/V5His
CMVp lacZ V5His
attB2
attB2 attB3attB1attB4
Invitrogen Proprietary & Confidential10
Effect of att sites on mammalian protein expression
* Extracts normalized for total protein
Gateway (G)
MultiSite CMV/ORFV5His/
CMVp ORF V5HisattB2 attB3attB1attB4
Non Gateway (NG)
pCDNA3.1 D-TOPO/ORF
ORFCMVp V5His
Invitrogen Proprietary & Confidential11
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
Invitrogen Proprietary & Confidential12
In silico cloning using Vector NTI AdvanceTM 10.3
Primers for PCR reaction
Cloning Strategy
DNA of interest
Invitrogen Proprietary & Confidential13
Your Application
Gene1 Gene2 Gene3 Gene4
Your Application
Gene
ProteinLocalization
Gene
Gene
ProteinPurification
Gene
RNAi
Gene
Cell-Free
Gene
Protein interaction
Gene
Gene
Entry Clone
PCR
Genesynthesis
ORFcollection
Library
YourSource
Gateway® Greatly Facilitates Protein Expression Studies
