Transcript Gene Expression - Life Technologies

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.
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
2
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Destination vectors for protein expression
E. coli
Native protein expression
Cell free
Mammalian
Insect
N-terminal fusion protein expression
Yeast
Virus
3
C-terminal fusion protein expression
Invitrogen Proprietary & Confidential
Examples of Destination vectors
For the expression of C-terminal
fusion or native proteins
4
For expression of N-terminal
fusion proteins
Invitrogen Proprietary & Confidential
Methods for Obtaining Gateway® Expression Clones
attR1
ccdB
attR2
attL1
+
Destination Vector
Gene
attL2
Entry Clone
LR Reaction
attB1
Gene
attB2
Expression Clone
TOPO® Reaction
attB1
attB2
TOPO® Expression
vector
5
+
Gene
PCR Product
Invitrogen Proprietary & Confidential
Transfer of the CAT Gene into Multiple Destination
Vectors
Destination Vector/Application
Colonies
Background
Analysis*
Native
15,000
0
4/4
6xHis-fusion
10,650
0
4/4
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
Native protein
7,800
15
4/4
6xHis
6,350
30
4/4
CMV-promoter
7,950
0
4/4
SFV
4,500
0
4/4
Tet-regulated promoter
6,350
0
4/4
E. coli
GST-fusion
Insect
Mammalian
* positives/total
6
Invitrogen Proprietary & Confidential
Effect of att sites on prokaryotic protein expression
pET300/NT-Dest
pET302 NT-His
MM = MagicMedia™
LB = Luria-Bertani medium + 1mM IPTG
7
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Effect of att sites on prokaryotic protein expression
8
Invitrogen Proprietary & Confidential
Effect of att sites on mammalian protein expression
1
2
3
1) pCDNA3.1/lacZ/V5His
CMVp
lacZ
V5His
LacZ
2) pDEST40/lacZ/V5His
CMVp
attB1
attB2 V5His
lacZ
3) MultiSite CMV/lacZ/V5His
attB4 CMVp
attB1
lacZ
attB2 V5His
attB3
Extracts normalized for total protein
Western blot probed with anti V5 antibody
9
Invitrogen Proprietary & Confidential
Effect of att sites on mammalian protein expression
Gateway (G)
MultiSite CMV/ORFV5His
/
attB4 CMVp
attB1
ORF
attB2 V5His
attB3
Non Gateway (NG)
pCDNA3.1 D-TOPO/ORF
* Extracts normalized for total protein
10
CMVp
ORF
V5His
Invitrogen Proprietary & Confidential
Gateway® Conversion Kit
R2
R1
Gateway® Cassette
+
R2
R1
Your destination vector
Promoter
Your linearized vector
Conversion cassettes can be used with any vector or
system, even the proprietary ones
11
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In silico cloning using Vector NTI AdvanceTM 10.3
DNA of
interest
Primers
for PCR
reaction
Cloning
Strategy
12
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Gateway® Greatly Facilitates Protein Expression Studies
PCR
Your
Source
Library
ORF
collection
Gene
synthesis
Gene
Gene
Gene
Protein
Localization
Your Application
Gene
Entry Clone
Gene
Gene
Protein
Purification
RNAi
Gene
Gene
Cell-Free
Protein
interaction
Gene1
Gene2
Gene3
Gene4
Your Application
13
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