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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 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 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

4 The Central Dogma DNA  RNA  Protein

5 The key steps in gene expression
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

6 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

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

9 Choosing an expression system
Protein Production Functional Analysis In vitro Bacteria Yeast Insect Mammalian Ease of Use Cost of media and equipment PTM / Probability of protein function This is a schematic to simplify the way we think about the traditional in vivo expression hosts. As most of you know, bacteria are a cheap and easy system to produce and purify protein. However, there may be a trade off in terms of protein function in using a bacterial system. Other protein production challenges protein solubility and activity. In vitro expression is included on this chart because, as you’ll see a bit later, this is a good option for addressing these challenges. If a protein requires post-translational modifications (PTM) for activity, then a bacterial system may not be optimal. As you move up the evolutionary ladder systems increase some in their difficulty to use, but you will get a payoff with more active protein. For cellular functional analysis, usually a mammalian system is used because most researchers want to study their protein in the context of the mammalian cellular environment. However, when a recombinant protein is produced and purified, there are many variables to consider in selecting a system. What we would like to do now is briefly go over a couple key variables and discuss some data with these host systems. Many people start out on a research pathway thinking that they’ll only use one system, but the truth is that often the need arises to move into different vectors within the same host system, or even move into multiple expression systems. Researchers may need use more than one system at any time as they pursue multiple approaches to studying a protein. Later I’ll talk about an easy way to rapidly move between different vectors and hosts. Time Requirement

10 Protein production-getting enough protein
Quantity (How much protein do you require?) ng μg mg g kg Mammalian Insect Yeast Bacterial in vitro How much protein will you need for downstream applications? Use the guide to your left to find the best system suitable for your needs.

11 Protein production - post-translational modifications?
Are PTMs required? No/Don’t Know In vitro (Expressway™ Plus) Bacterial (pET vectors) Pull down Interaction Studies Toxic (µgs) Structural (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? Express in Eukaryotic System The systems we’ve been talking about are primarily used for producing purified protein. Now we’ll move onto protein expression in a cellular context. Most people expressing protein in mammalian cells are doing so because there is a specific environment required for their experiments. Often that environment is a cell line that mimics a specific developmental or diseased state, or intact signaling pathways or specific protein partners are important for functional protein analysis. In optimizing mammalian experiments, a couple key questions must be considered: The best type of delivery Whether it will be helpful to be able to regulate gene expression using an inducible system

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

13 Get into any expression system with Gateway® Technology
Gene In Vitro Tags Your Vector Viral System Mammalian Baculovirus Yeast E. coli Entry Clone If you are interested in an in vitro system for cell-free expression, find out by visiting our Expressway™ seminar. We also have a special seminar for our Baculovirus BaculoDirect™ Expression system. For E. coli, yeast, mammalian, and viral expression systems, view our list of DEST vectors at Get into any expression system with Gateway® Technology

14 Gene Expression in E. coli

15 Optimization of Protein Expression
b-Gal GFP - + 6xHis Fusion2 6xHis-Trx Fusion E. coli strain BL21 SI (salt-inducible, T7 promoter) GUS GST Fusion1 Optimization of Protein Expression In this experiment, we cloned and expressed GUS, GFP, and B-gal with a GST or 6xHis fusion. As an example, we obtained higher levels of protein with the B-gal His fusion than with the GST fusion. In addition, the His tag can be cleaved off with the thioredoxin tag. 1 pDEST™ pDEST™17

16 Expression Vector Design
B2 B1 Apr Gene ATG Stop Promoter rbs Native Proteins Fusion Proteins 6xHis GST Myc V5 For native protein expression, your entry clone should contain an endogeneous start (ATG) and stop codon so that you won’t get any additional read through. If you want to express and either detect or purify your protein, use epitope tags. Invitrogen offers a variety of N- and C-terminal tags such as 6xHis for detection and purification, our Lumio™ tag for in-cell and in-gel detection, GST for purification, etc. When you are creating your Gateway® entry clone, see the diagram (at your left) or visit our online Vector Designer™ system.

17 Do attB Sites Affect Expression in E. coli?
topo pENTR SD/D-TOPO® att L1 RBS att L2 topo RBS ORF pET-DEST42 T7 lac O att B1 att B2 V5 6XHis vs. To determine whether the Gateway® attB sites affect protein expression for a panel of human kinase genes, the ORFs were cloned into the pENTR/SD/D-TOPO® entry vector. The entry clones were subsequently recombined into pET-DEST42. The ORFs were also directly cloned into pET101/D-TOPO®. pET-DEST42 is a Gateway® destination vector with the attR sites, and pET101/D-TOPO® does not have any att sites. ORF topo pET 101 D-TOPO® T7 lac O RBS V5 6XHis topo

18 Expression in Standard and Gateway®-Modified Vectors
GUS 6xHis-Gus GST-GUS Trx-GUS U I Std GW MW 50 kDa E. coli strain BL21-SI U = Uninduced, I = Induced The GUS gene was cloned using restriction enzymes and ligase (Std) into expression vectors or transferred via Gateway® Technology into destination vectors for native or fusion protein expression (GW). Expression clones generated via Gateway® Technology are flanked by attB sites. Induced cultures of BL21-SI™ containing these constructs show similar levels of protein expression suggesting the attB sites have no detectable effect on protein expression levels. What is the effect of Gateway® cloning on protein activity? We subcloned the GUS reporter gene into a mammalian expression vector, transfected cells, and then stained for activity.

19 Expression levels of Human Kinases
Expression levels for the panel of human kinases are expressed in arbitrary units. Overall, you can see the presence of att sites did not exhibit adverse affects on expression levels. *comparative expression levels are depicted in arbitrary units

20 Expression of Full-Length Human ORFs
Baculovirus/Sf9 Insect Cells 4 5 6 7 8 1 2 3 E. coli strain BL21 SI 20 50 220 Lane 1: 6xHis-GUS Lane 2: 6xHis-MAP4 Lane 3: 6xHis-b-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: b-Adaptin Lane 6: Transferrin Receptor Lane 7: Tyr Kinase Lane 8: EIF4e We have evaluated the expression of a number of full-length RT-PCR products in both E. coli and baculovirus expression systems. As you can see, not all proteins express in both systems, and the level of recombinant protein expressed can also differ. For example, we were able to get expression of human-EIF4e protein in E. coli (lane 6, gel on left, orange circle), but not in Sf9 cells (lane 8, gel on right, orange circle); the opposite was true for human-MAP4 (lane 2, left as compared to lane 4, right, green circles).

21 human p70 ribosomal S6 kinase
5 6 7 8 4 3 2 1 M u i pET-DEST42 pET 101 DT kDa 51 64 39 In this slide you will see data for the S6 Kinase ORF that was expressed in either pET-DEST42 or pET101-D-TOPO®. The ORF is expressed equally well in the Gateway® system. *58kd

22 Female sterile homeotic protein
5 6 7 8 4 3 2 1 M k Da 97 66 u i pET-DEST42 pET 101 DT In this slide you will see data for a homeotic protein that was expressed in either pET-DEST42 or pET101-D-TOPO®. The ORF is expressed equally well in the Gateway® system. *85 k Da protein

23 Receptor tyrosine kinase ligand
k Da M 1 2 3 4 5 6 7 8 31 21 In this slide you will see data for the Kinase liquid ORF that was expressed in either pET-DEST42 or pET101-D-TOPO®. For the receptor tyrosine kinase ligand, you may notice that the protein is slightly larger in the pET-DEST42 clone compared to the pET 101 DT clone. This difference in size is due to additional sequence from the pENTR/SD/D-TOPO® vector. The ORF is expressed equally well in the Gateway® system. u i u i u i u i pET 101 D-TOPO® Non-Gateway® pET DEST 42 Gateway® *23kd

24 Gene Expression Mammalian Cells

25 Two Entry Points for Expression
L1 L2 L1 L2 ORF pENTR/D-TOPO® pENTR-ORF R1 R2 pcDNA3.2-DEST V5 ccdB LR ORF There are two ways you can create an expression clone for high-level constitutive expression in mammalian cells. You can clone your gene of interest into a TOPO® entry vector (pENTR/D-TOPO®) and recombine into a destination vector. Or, you can clone directly into a Gateway®-compatible expression vector with TOPO® Cloning. You won’t have to perform any recombination reactions until you want to transfer your gene from the pcDNA™ vector to other vectors for further analysis. B1 B2 B1 B2 ORF V5 V5 pcDNA3.2 GW-ORF pcDNA3.2/GW/D-TOPO® *You can also clone your PCR product directly into this vector bypassing entry clone construction

26 pcDNA/GW/ D-TOPO® Vectors
Save cloning and screening time with pcDNA/GW/D-TOPO® expression vectors. Directional TOPO® provides fast, directional cloning via a 5-minute ligation of your PCR products. A built-in powerful CMV promoter drives high-level constitutive expression in a wide variety of cells. Gateway® recombination sites enable easy access to multiple downstream applications.

27 Expression of ORFs in CHO Cells
lacZ Gus GS5 GS10 GS15 GS19 A3 B9 GFP GS2 GS7 120kD 80kD 50kD 30kD Here we cloned various ORFs into the Gateway® pcDNA™ vector, transfected into Chinese Hamster Ovary cells (CHO), and used the V5 epitope antibody for detection. 20kD 1 2 3 4 5 6 7 8 9 10 11

28 Expression in Mammalian Cells
8 x 104 COS-7L cells, 0.8 mg each DNA/ well, 24 h post-transfection Lipofectamine™ 2000 Reagent (ml) 1.0 1.5 2.0 2.5 3.0 3.5 pCMVneo-GUS pCMV•SPORT-bgal We reacted the GUS Entry clone with the pCMVneo destination vector, and transfected COS-7L cells with the resulting expression clone. The control for this analysis was pCMV·SPORT-bgal DNA, our standard reporter plasmid. Following transfection, the cells were fixed and stained for reporter gene activity. As you can see, the Gateway® GUS-reporter plasmid was active. Let’s briefly take a look at the design of protein expression vectors, as their design is intimately related to that of the destination vector.

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