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Overview of Gene Expression Systems with Gateway ® Technology.

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Presentation on theme: "Overview of Gene Expression Systems with Gateway ® Technology."— Presentation transcript:

1 Overview of Gene Expression Systems with Gateway ® Technology

2 2 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

3 3 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

4 4 The Central Dogma DNA RNA Protein

5 5 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

6 6 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

7 7 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

8 8 Five primary systems used for expression… Mammalian Insect Yeast E. coli in vitro (aka cell free)

9 9 BacteriaYeastInsectMammalian Time Requirement Cost of media and equipment PTM / Probability of protein function Ease of Use Protein Production Functional Analysis In vitro Choosing an expression system

10 10 Protein production-getting enough protein Quantity (How much protein do you require?) ng μg mg g kg MammalianInsectYeastBacterialin vitro

11 11 Protein production - post-translational modifications? Are PTMs required? No/Dont Know In vitro (Expressway Plus) Bacterial (pET vectors) Pull down Interaction Studies Toxic (µgs) Structural Studies (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

12 12 Protein production - typical challenges Solubility (Do you have difficulty expressing your protein in bacteria?) Use fusions to improve solubilityTry a eukaryotic system

13 13 Gene In Vitro Tags Your Vector Viral System Mammalian Baculovirus Yeast E. coli Entry Clone Gene Get into any expression system with Gateway® Technology

14 14 Gene Expression in E. coli

15 15 Optimization of Protein Expression -Gal GFP -Gal xHis Fusion 2 6xHis-Trx Fusion E. coli strain BL21 SI (salt-inducible, T7 promoter) -Gal GUSGFP GST Fusion 1 1 pDEST15 2 pDEST17

16 16 Expression Vector Design B2B1 Ap r Gene ATGStop Promoter rbs Native Proteins B2B1 Ap r Gene ATGStop Promoter Fusion Proteins 6xHis GST Myc V5 rbs

17 17 Do attB Sites Affect Expression in E. coli? T7 lac ORBS topo V56XHis topo pET 101 D-TOPO® pET-DEST42 V56XHis att B2 T7lac Oatt B1 pENTR SD/D-TOPO® att L1RBS topo att L2 RBSORF vs.

18 18 Expression in Standard and Gateway®-Modified Vectors GUS6xHis-Gus GST-GUS Trx-GUS UIIIIUUU StdGWStdGWStdGWStdGW UIIIIUUU MW 50 kDa E. coli strain BL21-SI U = Uninduced, I = Induced

19 19 Expression levels of Human Kinases *comparative expression levels are depicted in arbitrary units

20 20 Expression of Full-Length Human ORFs Baculovirus/Sf9 Insect Cells E. coli strain BL21 SI Lane 1: 6xHis-GUS Lane 2: 6xHis-MAP4 Lane 3: 6xHis- -Adaptin Lane 4: 6xHis-Transferrin Receptor Lane 5: 6xHis-Tyr Kinase Lane 6: 6xHis-EIF4e kDa Lane 1: GST-GUS Lane 2: 6xHis-GUS Lane 3: GUS Lane 4: MAP4 Lane 5: -Adaptin Lane 6: Transferrin Receptor Lane 7: Tyr Kinase Lane 8: EIF4e

21 21 human p70 ribosomal S6 kinase MM uiuuuiii pET-DEST42 pET 101 DT kDa *58kd

22 22 Female sterile homeotic protein M k Da uuuuiiii pET-DEST42 pET 101 DT * 85 k Da protein

23 23 Receptor tyrosine kinase ligand M k Da uuuuiiii pET DEST 42 Gateway® pET 101 D-TOPO® Non-Gateway® *23kd

24 24 Gene Expression Mammalian Cells

25 25 Two Entry Points for Expression L1L2 pENTR/D-TOPO® B1B2 pcDNA3.2/GW/D-TOPO® ORF V5 ORF pENTR-ORF L1L2 R1R2 pcDNA3.2-DEST V5 ccdB B1B2 pcDNA3.2 GW-ORF V5 ORF LR *You can also clone your PCR product directly into this vector bypassing entry clone construction

26 26 pcDNA/GW/ D-TOPO® Vectors

27 27 Expression of ORFs in CHO Cells lacZGusGS5GS10GS15GS19A3 B9 GFPGS2GS7 120kD 80kD 20kD 30kD 50kD

28 28 8 x 10 4 COS-7L cells, 0.8 g each DNA/ well, 24 h post-transfection Lipofectamine 2000 Reagent ( l) pCMVneo-GUS pCMVSPORT- gal Expression in Mammalian Cells


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