Presentation on theme: "Overview of Gene Expression Systems with Gateway® Technology"— Presentation transcript:
1Overview of Gene Expression Systems with Gateway® Technology
2Research trendsCurrent research focuses on proteomics: drug target exampleApproximately 35,000 human genes in the genomeEstimated 500,000 targets for drug actionThe 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
3Gene expression areas of study Structural Proteomics - Protein ProductionMake lots of protein to use in other experimentsInteractionsStructureMake lots of protein to be used as a therapeutic (bioproduction)Functional Proteomics - Protein ExpressionStudy effects of protein expression in a cellIdentify the cellular functions of a proteinOver expression or gene knockdownStudy the function of a protein in different types of cells
5The key steps in gene expression Buy or Isolate GeneDetermine Expression SystemExpress Protein in CultureAnalyze the Recombinant ProteinTransfer gene into Expression VectorSelect gene and enter into chosen systemGenerate recombinant protein and analyze
6Generating Recombinant Protein - Overview See Getting intoGateway® slidesBuy or Isolate GeneDetermine Expression SystemTransfer gene into Expression VectorExpress Protein in CultureAnalyze the Recombinant Protein
7Generating Recombinant Protein - Overview Buy or Isolate GeneDetermine Expression SystemExpress Protein in CultureAnalyze the Recombinant ProteinTransfer gene into Expression VectorSelect gene and enter into chosen systemGenerate recombinant protein and analyze
8Five primary systems used for expression… InsectMammalianYeastin vitro(aka cell free)E. coli
9Choosing an expression system Protein ProductionFunctional AnalysisIn vitroBacteriaYeastInsectMammalianEase of UseCost of mediaand equipmentPTM / Probability of protein functionThis 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
10Protein production-getting enough protein Quantity (How much protein do you require?)ngμgmggkgMammalianInsectYeastBacterialin vitroHow much protein will you need for downstream applications?Use the guide to your left to find the best system suitable for your needs.
11Protein production - post-translational modifications? Are PTMs required?No/Don’t KnowIn vitro (Expressway™ Plus)Bacterial(pET vectors)Pull down InteractionStudiesToxic(µgs)Structural(mg to g)Structural studies; antigen production (mg to g)pull down studiesInsect (Baculodirect™)Mammalian(FreeStyle™)Yes-What kind of experiments are you doing?-How much protein do you need?Express in Eukaryotic SystemThe 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 deliveryWhether it will be helpful to be able to regulate gene expression using an inducible system
12Protein production - typical challenges Solubility (Do you have difficulty expressing your protein in bacteria?)Use fusions to improve solubilityTry a eukaryotic system
13Get into any expression system with Gateway® Technology GeneIn VitroTagsYour VectorViral SystemMammalianBaculovirusYeastE. coliEntry CloneIf 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 atGet into any expression system with Gateway® Technology
15Optimization of Protein Expression b-GalGFP-+6xHis Fusion26xHis-TrxFusionE. coli strain BL21 SI (salt-inducible, T7 promoter)GUSGST Fusion1Optimization of Protein ExpressionIn 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
16Expression Vector Design B2B1AprGeneATGStopPromoterrbsNativeProteinsFusion Proteins6xHisGSTMycV5For 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.
17Do attB Sites Affect Expression in E. coli? topopENTR SD/D-TOPO®att L1RBSatt L2topoRBSORFpET-DEST42T7lac Oatt B1att B2V56XHisvs.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.ORFtopopET 101 D-TOPO®T7lac ORBSV56XHistopo
18Expression in Standard and Gateway®-Modified Vectors GUS6xHis-GusGST-GUSTrx-GUSUIStdGWMW50 kDaE. coli strain BL21-SIU = Uninduced, I = InducedThe 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.
19Expression 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
20Expression of Full-Length Human ORFs Baculovirus/Sf9 Insect Cells45678123E. coli strain BL21 SI2050220Lane 1: 6xHis-GUSLane 2: 6xHis-MAP4Lane 3: 6xHis-b-AdaptinLane 4: 6xHis-Transferrin ReceptorLane 5: 6xHis-Tyr KinaseLane 6: 6xHis-EIF4ekDaLane 1: GST-GUSLane 2: 6xHis-GUSLane 3: GUSLane 4: MAP4Lane 5: b-AdaptinLane 6: Transferrin ReceptorLane 7: Tyr KinaseLane 8: EIF4eWe 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).
21human p70 ribosomal S6 kinase 56784321MuipET-DEST42pET 101 DTkDa516439In 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
22Female sterile homeotic protein 56784321Mk Da9766uipET-DEST42pET 101 DTIn 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
23Receptor tyrosine kinase ligand k DaM123456783121In 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.uiuiuiuipET 101 D-TOPO®Non-Gateway®pET DEST 42 Gateway®*23kd
25Two Entry Points for Expression L1L2L1L2ORFpENTR/D-TOPO®pENTR-ORFR1R2pcDNA3.2-DESTV5ccdBLRORFThere 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.B1B2B1B2ORFV5V5pcDNA3.2 GW-ORFpcDNA3.2/GW/D-TOPO®*You can also clone your PCR product directly into this vector bypassing entry clone construction
26pcDNA/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.
27Expression of ORFs in CHO Cells lacZGusGS5GS10GS15GS19A3B9GFPGS2GS7120kD80kD50kD30kDHere we cloned various ORFs into the Gateway® pcDNA™ vector, transfected into Chinese Hamster Ovary cells (CHO), and used the V5 epitope antibody for detection.20kD1234567891011
28Expression in Mammalian Cells 8 x 104 COS-7L cells, 0.8 mg each DNA/ well, 24 h post-transfectionLipofectamine™ 2000 Reagent (ml)1.01.52.02.53.03.5pCMVneo-GUSpCMV•SPORT-bgalWe 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.