Overview of Gene Expression Systems with Gateway ® Technology.

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 targets…need to express to characterize

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• 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

Gene expression areas of study

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The Central Dogma

DNA RNA Protein

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Buy or Isolate Gene

Determine Expression System

Express Protein in Culture

Analyze the Recombinant Protein

Transfer gene into Expression Vector

Select gene and enter into chosen system

Generate recombinant protein and analyze

The key steps in gene expression

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Generating Recombinant Protein - Overview

Buy or Isolate Gene

Determine Expression System

Express Protein in Culture

Analyze the Recombinant Protein

Transfer gene into Expression Vector

See Getting into

Gateway® slides

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Generating Recombinant Protein - Overview

Buy or Isolate Gene

Determine Expression System

Express Protein in Culture

Analyze the Recombinant Protein

Transfer gene into Expression Vector

Select gene and enter into chosen system

Generate recombinant protein and analyze

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Five primary systems used for expression…

Mammalian

Insect

Yeast

E. coli

in vitro

(aka cell free)

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Bacteria Yeast Insect Mammalian

Time Requirement

Cost of mediaand equipment

PTM / Probability of protein function

Ease of Use

Protein Production

Functional Analysis

In vitro

Choosing an expression system

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Protein production-getting enough protein

• Quantity (How much protein do you require?)

ng

μg

mg

g

kg

MammalianInsectYeastBacterial in vitro

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Protein production - post-translational modifications?

Are PTMs required?

No/Don’t Know

In vitro (Expressway™

Plus)

Bacterial (pET vectors)

Pull down InteractionStudiesToxic(µgs)

StructuralStudies

(mg to g)

Structural studies; antigen production

(mg to g)

pull down studies

Insect (Baculodirect™)

Mammalian(FreeStyle™)

Yes

-What kind of experiments are you doing?-How much protein do you need?

-What kind of experiments are you doing?-How much protein do you need?

Express in Eukaryotic System

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Protein production - typical challenges

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

Use fusions to improve solubility Try a eukaryotic system

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Gene

Gene

Gene

Gene

Gene

Gene

GeneGene

In Vitro

Tags

Your Vector

Viral System

Mammalian

Baculovirus

Yeast

E. coli Entry Clone

Gene

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

-Gal GFP -Gal

- + - + - + - + - + - +

6xHis Fusion2

6xHis-TrxFusion

E. coli strain BL21 SI (salt-inducible, T7 promoter)

-GalGUS GFP

- + - + - + - + - + - +

GST Fusion1

1 pDEST™15 2 pDEST™17

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Expression Vector Design

B2B1

Apr

Gene

ATG Stop

Promoter

rbs

Native Proteins

B2B1

Apr

Gene

ATG Stop

Promoter

Fusion Proteins

6xHisGST

MycV5 rbs

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Do attB Sites Affect Expression in E. coli?

T7 lac O

RBS

topo

V5 6XHis

topopET 101 D-TOPO®

pET-DEST42 V5 6XHisatt B2

T7 lac O

att B1

pENTR SD/D-TOPO® att L1 RBS

topo

topo

att L2

RBS ORF

ORF

vs.

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Expression in Standard and Gateway®-Modified Vectors

GUS 6xHis-Gus GST-GUSTrx-GUS

U II I IU UU

Std GWStdGW Std GWStdGW

U II I IU UU

MW MW MW MW

50 kDa

E. coli strain BL21-SI U = Uninduced, I = Induced

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Expression levels of Human Kinases

ORF MW (kDa) pET-DEST42* pET 101 DT*H-U27143 14 + ++H-L25610 18 ++ ++H-M57730 23 +++ +H-X79483 37 - -H-D87116 38 ++ ++H-U02680 39 ++ +H-U00803 56 ++ ++H-M36881 56 ++ -H-M60724 58 ++ +++H-X52479 82 + +H-M80613 83 + +H-X97335 99 + +

*comparative expression levels are depicted in arbitrary units

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Expression of Full-Length Human ORFs

Baculovirus/Sf9 Insect Cells

4 5 6 7 81 2 3

E. coli strain BL21 SI

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50

220

1 2 3 4 5 6

Lane 1: 6xHis-GUSLane 2: 6xHis-MAP4Lane 3: 6xHis--AdaptinLane 4: 6xHis-Transferrin ReceptorLane 5: 6xHis-Tyr KinaseLane 6: 6xHis-EIF4e

kDa

Lane 1: GST-GUSLane 2: 6xHis-GUS Lane 3: GUSLane 4: MAP4Lane 5: -AdaptinLane 6: Transferrin ReceptorLane 7: Tyr Kinase Lane 8: EIF4e

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human p70 ribosomal S6 kinase

5 6 7 84321M M

u i u u ui i i

pET-DEST42 pET 101 DT

kDa

51

64

39

*58kd

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Female sterile homeotic protein

5 6 7 84321Mk Da

97

66

u u u ui i i i

pET-DEST42 pET 101 DT

*85 k Da protein

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Receptor tyrosine kinase ligand

5 6 7 84321Mk Da

u u u ui i i i

pET DEST 42 Gateway®

pET 101 D-TOPO®Non-Gateway®

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21

*23kd

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Gene Expression Mammalian Cells

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Two Entry Points for Expression

L1 L2

pENTR/D-TOPO®

B1 B2

pcDNA3.2/GW/D-TOPO®

ORF

V5

ORF

pENTR-ORF

L1 L2

R1 R2

pcDNA3.2-DEST

V5ccdB

B1 B2

pcDNA3.2 GW-ORF

V5ORF

LR

*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 GS1

0 GS1

5 GS1

9 A3 B9

GFP GS2 GS7

120kD

80kD

20kD

30kD

50kD

1 2 3 4 5 6 7 8 9 10 11

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8 x 104 COS-7L cells, 0.8 g each DNA/ well, 24 h post-transfection

Lipofectamine™ 2000 Reagent (l)

1.0 1.5 2.0 2.5 3.0 3.5

pCMVneo-GUS

pCMV•SPORT-gal

Expression in Mammalian Cells