Cell-Free (In Vitro) Protein Expression - How It Helps Speed Up Your Research

Transcription

1 Cell-Free (In Vitro) Protein Expression - How It Helps Speed Up Your Research Gary Kobs January 14, Proprietary Information. Not for further distribution.

2 Presentation Overview Cell-Free (In Vitro) Protein Expression Overview Faster, easier protein expression with different systems to match your protein expression needs Non-radioactive labeling and detection of expressed proteins Key advantages of each system with data examples Applications of Cell-Free Expressed Proteins Protein:protein interaction studies Protein:DNA binding studies Assessment of viral IRES function Structural studies Ubiquitination analysis Analysis of membrane proteins Drug screening HaloTag Technology Overview A unique, multi-functional protein fusion tag for global protein characterization Combined Usage of HaloTag and Cell-Free Expressed Proteins Protein kinase assays and inhibitor screen Summary Proprietary Information. Not for further distribution. 2

3 Easiest/Fastest Method from DNA to Protein Saves valuable time Cell-free competent plasmid or Cell-free competent PCR product Template TNT System Master Mix C Label - None - 35 S-methionine - Biotinylated lysines - Fluor-labeled lysines T7 RNA polymerase Cellular lysate Nucleotides Amino acids Energy regenerating system Transcription/translation Protein of Interest Produce protein in 1-2 hours vs. days to weeks in E. coli or mammalian cells Use PCR product templates and bypass cloning ORF into expression vector Produces sufficient protein for many applications including: Protein:protein interactions Co-IPs, pulldowns Protein:nucleic acid interactions Gel shift assays Enzymatic assays Enables analysis of difficult proteins Normally toxic to cells Insoluble in E. coli Simplifies detection Directly label protein during synthesis Multiple Applications Fluorophore, 35 S, biotin Proprietary Information. Not for further distribution. 3

4 Choices to Match Your Research Needs Prokaryotic Eukaryotic Bacteria Plant Insect Mammalian E. coli Wheat germ Spodoptera frugiperda Rabbit reticulocyte S30 T7 High-Yield Protein Expression System TNT SP6 High-Yield Wheat Germ System TNT T7 Insect Cell Extract System TNT Quick Coupled T7 and SP6 Systems Highest yield Maximal soluble protein Most active protein Native mammalian system Proprietary Information. Not for further distribution. 4

5 Faster Protein Production than E. coli Systems Vector Prep E. coli Expression Analysis or Purification Template Prep TNT System Analysis or Purification 2 hrs Critical Timeline 2 days Proprietary Information. Not for further distribution. 5

6 Maximal Yields from Each System S30 T7 High-Yield Protein Expression System TNT SP6 High-Yield WG System TNT T7 Insect Cell System dialysis TNT Quick Protein Yield (µg/ml) Proprietary Information. Not for further distribution. 6

7 Characteristics of Cell-Free Expression Systems System Time Yield Requirements TNT T7 or SP6 Quick Coupled System (RRL) S30 High Yield (Bacterial) TNT SP6 High-Yield Wheat Germ System TNT T7 Insect Cell System 1 hour 0.5µg/50µl 1 hour 25µg/50µl 2 hours µg/50µl 4 hours 4.0µg/50µl Any vector containing a T7 or SP6 promoter upstream of coding sequence. T7 promoter-driven bacterial expression vector. Can also use very active bacterial promoters (T5). Highest yield with specialized vector containing plant viral sequences (5.0µg/rxn). Greatest yield using dialysis method (12.5µg/rxn). Require use of a baculovirus expression vector with T7 promoter upstream. Optimized Wheat Germ Vector Insect Cell Vector Example Proprietary Information. Not for further distribution. 7

8 Protein 1 Protein 2 Protein 3 Protein 4 Protein 5 Lysate only Fluorescent Detection of Expressed Proteins Non-Radioactive Co-translational Labeling FluoroTect Green Lys trna TNT T7 Quick Coupled System 250kDa 150kDa 100kDa 75kDa 102 kda 76 kda 52 kda 50kDa 38 kda 37kDa Proteins expressed TNTT7 Quick Coupled system TNT S 250kDa 150kDa 100kDa 75kDa 50kDa 37kDa lys lys Translation reaction + FluoroTect Green Lys trna lys FluoroTect Green 24 kda 25kDa 17 kda Lysate Only Proteins 25kDa Produce active/detectable proteins without radioactivity Direct detection of fluorescently labeled proteins in gels Use in many applications including pulldowns, co-immunoprecipitations, mobility shift assays Proprietary Information. Not for further distribution. 8

9 Protein 1 Protein 2 Protein 3 Protein 4 Protein 5 Lysate only Protein 1 Protein 2 Protein 3 Protein 4 Protein 5 Lysate only Indirect Detection of In Vitro Expressed Proteins Transcend Biotin Co-translational Labeling lys 102 kda 76 kda 52 kda 38 kda 24 kda 17 kda lys Proteins expressed TNTT7 Quick Coupled system Transcend Biotinylated trna Translation reaction + Transcend Biotinylated trna lys Biotin TNT SP6 Wheat Germ High-Yield System Proteins expressed TNT SP6 High Yield Wheat Germ system 102 kda 102 kda 76 kda 52 kda 76 kda 52 kda 38 kda 38 kda 24 kda 24 kda 17 kda 17 kda Lysate Only Proteins Produce active/detectable proteins without radioactivity Indirect detection of biotin labeled proteins using streptavidin conjugates (HRP, AlkPhos) Use in many applications including pull downs, co-immunoprecipitations, mobility shift assays Proprietary Information. Not for further distribution. 9

10 Detection Sensitivity Rivaling 35 S-Methionine 5µl of reaction Direct in-gel detection Fluorescent 1µl of reaction Chemiluminescent Detection Biotin, Indirect 20µCi 35 S-Met 1µl of reaction overnight exposure Varying micrograms of firefly luciferase RNA were expressed in the Rabbit Reticulocyte Lysate System, Nuclease Treated (Cat. #L4960). Full details in Kobs, G., et al. (2001) Promega Notes 77, Radioactive Proprietary Information. Not for further distribution. 10

11 What if There is a Lysine in the Active Site? No Problem Only 25-33% of Lysines are Labeled FluoroTect & Transcend trna compete with natural lysyl trna for incorporation into growing peptide chain Lysine Lysine + Label 25%-33% of lysines get labeled UUU UUU Precharged Transcend or FluoroTect trna AA A / G Proprietary Information. Not for further distribution. 11

12 E. coli S30 Cell-Free Systems High Yield System Amenable to HTS Applications Bacteria Key Advantages Highly efficient expression = high yield system Great screening system Fast, suitable for HTS applications Open system that allows inclusion of additives Reported correlation with E. coli cell-based expression Well suited to protein labeling and large-scale production S30 T7 High-Yield Protein Expression System Documented success in membrane protein production General Vector/Template Requirements T7 vectors for E. coli cell-based expression Specialized vectors available Proprietary Information. Not for further distribution. 12

13 E. coli S30 Cell-Free Systems Protein Expression/Purification Manual MagneHis Purification Fluorescent Detection pfn6a-mggfp Clone Cell-Free E. coli Expression HQ Monster Green GFP S30 T7 High Yield System Automated 1-16 Sample Purification (Maxwell 16) Proprietary Information. Not for further distribution. 13

14 Wheat Germ Cell-Free Systems More Soluble Protein from an Animal-Free System Plant Key Advantages Full-length, soluble mammalian protein expression High yield system (200µg/ml) Suitable for structural biology applications Animal-free, eukaryotic system General Vector/Template Requirements TNT SP6 High-Yield Wheat Germ System SP6 or T7 promoter vectors depending on WG kit Specialized vectors with plant viral 5, 3 UTRs for higher expression available Proprietary Information. Not for further distribution. 14

15 Wheat Germ Cell-Free Systems More Soluble Protein from an Animal-Free System 55/55 Proteins Expressed in TNT SP6 High Yield WG System 55/55 soluble in WG System! Only 6/55 soluble in E. coli cellbased expression system Proprietary Information. Not for further distribution. 15

16 Insect Cell-Free Systems Highly Active Protein from an Animal-Free System Insect Key Advantages Production of highly active, full-length proteins Relatively high protein yields 75µg/ml in TNT coupled system Animal-free, eukaryotic system TNT T7 Insect Cell Extract System General Vector/Template Requirements T7 promoter vectors Specialized vectors available with baculovirus 5, 3 UTRs for higher expression Proprietary Information. Not for further distribution. 16

17 Fold (specific activiy/tntr) ng/ul Insect Cell-Free Systems Highly Active Protein from an Animal-Free System Highest Specific Activity cpka with TNT T7 Insect Cell System WG RR ICE no camp camp Protein Yield (WG>ICE>RR) Specific Activity (ICE>RR WG) Tested: TNT SP6 High Yield Wheat Germ System TNT T7 Quick (Rabbit Retic.) System TNT T7 Insect Cell Extract System 0 WG RR ICE Proprietary Information. Not for further distribution. 17

18 Rabbit Retic (Mammalian) Cell-Free Systems Mammalian Protein Expression in a Native Setting Mammalian Key Advantages Native system for mammalian proteins Post-translational processing Adding canine microsomal membranes Full-length protein expression likely TNT Quick Coupled T7 and SP6 Systems General Vector/Template Requirements T7 or SP6 promoter vectors T7 TNT System: Compatible with many templates A Universal Lysate Proprietary Information. Not for further distribution. 18

19 Rabbit Retic (Mammalian) Cell-Free Systems Universal Lysate Suitable for Many T7 Vectors T7 Vector Compatibility with Multiple T7 Expression Vectors Original Use Elements TNT Expression Possible? pf1a E. coli T7p-lacO-rbs-T7t Y pet15b-ffluc E. coli T7p-lacO-rbs-HisTag-T7t Y pet32a E. coli T7p-lacO-rbs-Trx-T7t Y pet43.1a E. coli T7p-lacO-rbs-NusA-T7t Y pivex S30 cell-free system T7p-lacO-rbs-T7t Y pix3.0 S30 cell-free system T7p-lacO-rbs-T7t Y pix4.0 Insect cell-free extract T7p- Y pf3k Wheat germ extract T7p-Sp6p-BYDV5 -BYDV-3 -T7t Y pfn19k Cell-free extracts T7p-Sp6p-N-HT7-polyA Y pfc20k Cell-free extracts T7p-Sp6p-C-HT7-polyA Y pf4a Mammalian CMV-intron-T7p-SV40late polya Y prl-null Mammalian T7p-SV40late polya Y prl-cmv Mammalian CMV-T7p-SV40late polya Y prl-sv40 Mammalian SV40-T7p-SV40late polya Y prl-tk Mammalian TK-T7p-SV40late polya Y Proprietary Information. Not for further distribution. 19

20 RLU Rabbit Retic (Mammalian) Cell-Free Systems Rapid Protein Expression/Functional Screening Easily Test Expression Vectors Containing T7 Promoters w/tnt T7 Quick pet15b T7p laco rbs His Renilla luciferase T7t pf1a T7p laco rbs Renilla luciferase T7t pf4a CMVp Intron T7p Renilla luciferase SV40 Late PolyA Signal Renilla Expression Renilla Luciferase Activity control pet15b pf1a pf4a Proprietary Information. Not for further distribution. 20

21 Cell-Free Technology and Detection Summary Quickly Express Proteins in the System of Your Choice Go from DNA to usable protein in 1-4 hours Overcomes toxicity and insolubility issues associated with many proteins Multiple cell-free systems available to match your needs E. coli >>> Maximal protein yield Wheat Germ >>> Most soluble protein Insect Cells >>> Functionally active protein Rabbit Reticulocyte >>> Native mammalian system Simplify detection with co-translational biotin or fluorescent labeling Indirect detection or direct fluorescent detection Radioactive labeling with 35 S-Met Western blotting detection using primary antibody to the expressed protein Proprietary Information. Not for further distribution. 21

22 Presentation Overview Cell-Free (In Vitro) Protein Expression Overview Faster, easier protein expression with different systems to match your protein expression needs Non-radioactive labeling and detection of expressed proteins Key advantages of each system with data examples Applications of Cell-Free Expressed Proteins Protein:protein interaction studies Protein:DNA binding studies Assessment of viral IRES function Structural studies Ubiquitination analysis Analysis of membrane proteins Drug screening HaloTag Technology Overview A unique, multi-functional protein fusion tag for global protein characterization Combined Usage of HaloTag and Cell-Free Expressed Proteins Protein kinase assays and inhibitor screen Proprietary Information. Not for further distribution. 22

23 Protein:Protein Interactions: Testing for Direct Interaction of Asr1 with the RNAP II CTD Daulny A., et al. (2008) Proc. Nat. Acad. Sci. 105, Key Experiments Using Cell-free Expressed Proteins Test for direct binding of Asr1 to the RNA Polymerase II CTD Analyze role of CTD phosphorylation in the interaction Used a cell-free expressed Asr1 protein in a Far Western assay to investigate the interaction Proprietary Information. Not for further distribution. 23

24 Protein:Protein Interactions: Testing for Direct Interaction of Asr1 with the RNAP II CTD Experimental Design Using Far Westerns to Detect Interactions Purified GST, GST-CTD WT & Ser2 & 5 mutant proteins GST PO 4 E. coli +/- Phosphorylation SDS-PAGE & transfer to membrane Incubate Wash Detect bound Asr1 by autoradiography 1 hour + 35 S-Met 35 S Asr1 TNT T7 Quick Coupled System Proprietary Information. Not for further distribution. 24

25 Protein:Protein Interactions: Testing for Direct Interaction of Asr1 with the RNAP II CTD Results 1. Asr1 binds directly to the RNAP II CTD in a phosphorylation dependent manner 2. Mutation of Ser5 to alanine prevents phosphorylation at Ala5 & blocks Asr1 binding 3. Phosphorylation of Ser2 plays less of a role in promoting Asr1 binding GST-CTD Fusion Protein: CTD Phosphorylation: Far Western Blot Coomassie Stained Gel (GSTΔ, GST-CTD Proteins) A2 = Ser2 to Ala mutation A5 = Ser5 to Ala mutation Daulny A., et al. (2008) Proc. Nat. Acad. Sci. 105, Proprietary Information. Not for further distribution. 25

26 Protein:DNA Binding and Protein:Protein Interaction Analysis of AHRR1 Protein Evans B., et al. (2008) Mol. Pharm. 73, Key Experiments Using Cell-free Expressed Proteins Analyze DNA binding of AHHR1 (aryl hydrocarbon receptor repressor) Illustrate that a point mutation abolishes DNA binding activity Test and analyze the interaction of AHHR1 with ARNT2 using CoIP assay Proprietary Information. Not for further distribution. 26

27 Protein:DNA Binding: Testing the DNA Binding Activity of AHHR1 and ARNT2 Experimental Design - Gel Shift Assays using Cell-Free Expressed Proteins TNT T7 Quick Coupled System 1 hour unlabeled AHRR1 Wild Type Y9F Mutant 32 P Mix & Incubate 32 P AHRR1 ARNT2 1 hour Run in a nondenaturing ARNT2 unlabeled polyacrylamide gel & autoradiograph Proprietary Information. Not for further distribution. 27

28 Protein:DNA Binding: Testing for AHHR1 DNA Binding Activity Results 1. AHRR1 binds the AHRE DNA binding site only in the presence of ARNT2b 2. Mutation of Tyr9 to Phe abolishes DNA binding 3. Supershift of the complex with anti-ahhr1 Ab demonstrates AHRR1 is in the complex Evans B., et al. (2008) Mol. Pharm. 73, Proprietary Information. Not for further distribution. 28

29 Protein:Protein Interactions: Testing for Interaction of AHHR1 and ARNT2 Co-immunoprecipitation Assay Expressed full-length and deletion mutants of AHRR1 using TNT T7 Quick-Coupled Rabbit Reticulocyte Lysate System AHHR1 FL & Deletions + 35 S-Met ARNT2 35 S-met labeled AHRR1 proteins Expressed ARNT2 protein using same system unlabeled Used anti-arnt2 antibody in CoIP protein complexes Detected Co-IP d AHRR1 proteins by SDS-gel & fluorography Proprietary Information. Not for further distribution. 29

30 Protein:Protein Interaction: Testing for Interaction of AHHR1 and ARNT2 Results Co-immunoprecipitation of AHRR1 and ARNT2 1. AHRR1 & ARNT2 interact to form a complex 2. Amino acids are required for the interaction Evans B., et al. (2008) Mol. Pharm. 73, Proprietary Information. Not for further distribution. 30

31 In Vitro Functional Analysis of the CrPV IRES Garrey J., et al. (2010) J. Virol. 84, Key Experiments Using Cell-free Expressed Proteins In vitro translation with dicistronic RNA containing viral IRES elements Used in vitro system to alter translation machinery and measure effects Transcribed/translated an additional protein and measured effect on IRES function Also used Dual Luciferase assay to measure levels of cell-free expression Proprietary Information. Not for further distribution. 31

32 In Vitro Functional Analysis of the CrPV IRES Experimental Design In Vitro Translation Assays using Cell-Free Expression System +/- 4E-BP1 Expression Plasmid +/- IRES (CrPV or IGR) RNA R luc FF luc Dual-Luciferase Assays R Luc + FF Luc TNT T7 Quick Coupled System 30 min + 35 S-Met 4E-BP1 0, 30, 60 min time points SDS gel followed by autoradiography Proprietary Information. Not for further distribution. 32

33 In Vitro Functional Analysis of the CrPV IRES Results 1. Both IRES s promote translation of FF luc in the second cistron In Vitro Translation Data 2. The IGR IRES is much more active than the CrPV IRES (5 UTR IRES) 3. 4E-BP1 disrupts scanning-mediated translation promoting IRES-mediated translation Garrey J., et al. (2010) J. Virol. 84, Proprietary Information. Not for further distribution. 33

34 Generation of Labeled Proteins for Structural Analysis by Solution NMR Zhao, Li et al. (2010) Struc.Funct.Gen.11, Key Experiments Using Cell-free Expressed Proteins Comparison of 59 human proteins for solubility and expression levels using wheat germ extract or E. coli Scaled expression for 2 labeled proteins followed by NMR analysis Proprietary Information. Not for further distribution. 34

35 Use of Cell-Free for Structural Studies Results 1. Only 30% of proteins produced soluble form when using E.coli 2. 70% of proteins produced soluble form when using wheat germ Proprietary Information. Not for further distribution. 35

36 Ubiquitination Analysis of p73 Suppressor Protein Jung, Y-S et al. (2011) J. Biol. Chem. 286, Key Experiments Using Cell-free Expressed Proteins In vitro translation of p73 with wild type and mutated Pirh2 in the presence of E1, E2 and ubiquitin Proprietary Information. Not for further distribution. 36

37 Use of Cell-Free for Ubiquitination Analysis Results 1. 1PirH2 physically interacts with p73 2. Pirh2 promotes p73 polyubiquitination Proprietary Information. Not for further distribution. 37

38 Membrane Association of Pestivirus Glycoprotein E rns Tews, B-A. et al. (2007) J. Biol. Chem. 282, Key Experiments Using Cell-free Expressed Proteins In vitro translation of wild type E rns and E rns containing mutations Proteinase K protection assays in the presence of canine microsomal membranes Proprietary Information. Not for further distribution. 38

39 Membrane Association of Pestivirus Glycoprotein E rns Results 1. Proteinase K protection assays showed show that E rns translated in the presence of microsomal membranes was protected 2. A mutant version containing a artificial transmembrane region and short cytosolic tail was shortened by protease treatment Proprietary Information. Not for further distribution. 39

40 Characterizing Toxins: E. coli YoeB Key Experiments Using Cell-free Expressed Proteins Monitoring the effect toxins YoeB and MazF on protein synthesis in both prokaryotic and eukaryotic cell-free systems Determine if the antitoxin YefM inhibited YeoB activity Proprietary Information. Not for further distribution. 40

41 Use of Cell-Free Expression for Screening Potential Toxins Results 1. YefM antitoxin mediated YoeB inhibition 2. YoeB is a specific inhibitor to only prokaryotic protein synthesis Proprietary Information. Not for further distribution. 41

42 Presentation Overview Cell-Free (In Vitro) Protein Expression Overview Faster, easier protein expression with different systems to match your protein expression needs Non-radioactive labeling and detection of expressed proteins Key advantages of each system with data examples Applications of Cell-Free Expressed Proteins Protein: protein interaction studies Protein-DNA binding studies Assessment of viral IRES function HaloTag Technology Overview A unique, multi-functional protein fusion tag for global protein characterization Combined Usage of HaloTag and Cell-Free Expressed Proteins Protein kinase assays and inhibitor screen Summary Proprietary Information. Not for further distribution. 42

43 What is HaloTag Technology? A Unique, Multifunctional Protein Fusion Tag HaloTag : Engineered 34.1kDa halophilic bacterial hydrolase Binds to chloralkane substrate and locks with covalent attachment Faster kinetics than the biotin:streptavidin interaction No homolog in mammalian cells = no background Read more about the development of this powerful fusion tag: Ohana, R.F., et al. (2009) Prot. Exp. Purif. 68, Protein of Interest HaloTag + N- or C- terminal fusions Protein of Interest Cl Chloroalkane Linker Binding and O O O O Functional Group Functional Group Covalent bond between HaloTag and chloroalkane (+ functional group) Proprietary Information. Not for further distribution. 43

44 Many Functional Groups are Available to Match Your Research Application(s) Protein of Interest O O Functional Group Cl O O HaloTag Surfaces/Resins Capture and Display Protein arrays Purification Interaction analysis Cl Cl O O O O HaloTag Fluorescent Ligands Labeling and Detection Cellular imaging Gel analysis Quantitation Cl O O HaloTag Reactive Ligands Custom Modifications Attach to particles, surfaces Attach special ligands Proprietary Information. Not for further distribution. 44

45 Presentation Overview Cell-Free (In Vitro) Protein Expression Overview Faster, easier protein expression with different systems to match your protein expression needs Non-radioactive labeling and detection of expressed proteins Key advantages of each system with data examples Applications of Cell-Free Expressed Proteins Protein: protein interaction studies Protein-DNA binding studies Assessment of viral IRES function HaloTag Technology Overview A unique, multi-functional protein fusion tag for global protein characterization Combined Usage of HaloTag and Cell-Free Expressed Proteins Protein kinase assays and inhibitor screen Summary Proprietary Information. Not for further distribution. 45

46 Enzymatic Assays Measuring Protein Kinase A (PKA) Activity Experimental Design Immobilization of HaloTag -cpka followed by Kinase Assays HT-cPKA Expression Plasmid TNT SP6 High Yield Wheat Germ System Incubate HaloTag HaloLink Magnetic Beads Cl cpka O O Purify Immobilized Enzyme HaloLink Array Slide cpka O O Kinase Assay ATP O O ADP O O Measure Remaining ATP O O Kinase-Glo PKA Kinase Assay ProFluor PKA Activity PKA Activity Proprietary Information. Not for further distribution. 46

47 Percent Maximum Signal (Inhibition) Percent Maximum Signal (Inhibition) Enzymatic Assays: Testing for PKA Inhibitors cpka O O WGE PKA Inhibitor Screening Compound Target cpka Inhibition U0126 MEK1/2 No H89 PKA specific Yes PKI PKA specific Yes Staurosporine DMSO +/- Inhibitors ProFluor Kinase Assay Higher Bars = More Inhibition ICE Non-selective Kinase inhibitor Yes No Donna M. Leippe, Kate Qin Zhao, Kevin Hsiao and Michael R. Slater. Analytical Chemistry Insights (2010)5, PKA Inhibitor Results 20 ul particles 10 ul particles 0.3 u rpka u rpka U0126 H89 PKI st aurosporine DMSO Buf f er Inhibitor (10 µm) 20 ul particles 10 ul particles 0.3 u rpka u rpka U0126 H89 PKI st aurosporine DMSO Buf f er Inhibitor (10 µm) WGE ICE Proprietary Information. Not for further distribution. 47

48 Summary In vitro (cell-free) expression is an easy-to-use system to rapidly produce full-length or deletion proteins of interest. Multiple cell-free systems are available to match your downstream requirements. Cell-free expressed proteins are suitable for a wide array of downstream applications. Protein:protein interaction studies, functional assays (e.g. kinase), protein: nucleic acid binding only limited by your creativity The combination of cell-free expression and HaloTag is a powerful tool for protein analysis. HaloTag provides a multi-functional tag for in vivo analysis as well Protein interaction studies, cellular localization and trafficking, protein:nucleic acid interactions Proprietary Information. Not for further distribution. 48