Overview of Gene Expression Systems with Gateway

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Overview of Gene Expression Systems with Gateway
Technology
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Research trends

• Current research focuses on proteomics drug
  target example
• Approximately 35,000 human genes in the genome
• Estimated 500,000 targets for drug action
• The correlation between gene expression and
  protein expression is less than 10
• Proteins need to be characterized in order to
  identify drug targetsneed to express to
  characterize

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Gene expression areas of study

• Structural Proteomics - Protein Production
• Make lots of protein to use in other experiments
• Interactions
• Structure
• Make lots of protein to be used as a therapeutic
  (bioproduction)
• Functional Proteomics - Protein Expression
• Study effects of protein expression in a cell
• Identify the cellular functions of a protein
• Over expression or gene knockdown
• Study the function of a protein in different
  types of cells

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The Central Dogma
DNA ? RNA ? Protein
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The key steps in gene expression
Select gene and enter into chosen system
Generate recombinant protein and analyze
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Generating Recombinant Protein - Overview
See Getting into Gateway slides
Buy or Isolate Gene
Determine Expression System
Transfer gene into Expression Vector
Express Protein in Culture
Analyze the Recombinant Protein
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Generating Recombinant Protein - Overview
Select gene and enter into chosen system
Generate recombinant protein and analyze
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Five primary systems used for expression
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Choosing an expression system
Bacteria
Yeast
Insect
Mammalian
In vitro
Ease of Use
Cost of media and equipment
PTM / Probability of protein function
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Protein production-getting enough protein
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Protein production - post-translational
modifications?
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Protein production - typical challenges

• Solubility (Do you have difficulty expressing
  your protein in bacteria?)

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Get into any expression system with Gateway
Technology
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Gene Expression in E. coli
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Optimization of Protein Expression
1 pDEST15 2 pDEST17
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Expression Vector Design
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Do attB Sites Affect Expression in E. coli?
topo
pENTR SD/D-TOPO
att L1
RBS
att L2
topo
RBS
ORF
pET-DEST42
att B2
V5
6XHis
T7
lac O
att B1
vs.
topo
pET 101 D-TOPO
T7
lac O
RBS
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Expression in Standard and Gateway-Modified
Vectors
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Expression levels of Human Kinases
comparative expression levels are depicted in
arbitrary units
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Expression of Full-Length Human ORFs
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human p70 ribosomal S6 kinase
58kd
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Female sterile homeotic protein
85 k Da protein
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Receptor tyrosine kinase ligand
5
4
3
2
1
M
6
7
8
k Da
31
21
u
u
u
u
i
i
i
i
pET 101 D-TOPO Non-Gateway
pET DEST 42 Gateway
23kd
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Gene Expression Mammalian Cells
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Two Entry Points for Expression
L1
L2
L1
L2
ORF
pENTR-ORF
pENTR/D-TOPO
LR
ORF
B1
B2
B1
B2
ORF
pcDNA3.2 GW-ORF
pcDNA3.2/GW/D-TOPO
You can also clone your PCR product directly
into this vector bypassing entry clone
construction
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pcDNA/GW/ D-TOPO Vectors
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Expression of ORFs in CHO Cells
lacZ
Gus
GS5
GS10
GS15
GS19
A3
GFP
GS2
GS7
B9
120kD
80kD
50kD
30kD
20kD
1
2
3
4
5
6
7
8
9
10
11
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Expression in Mammalian Cells