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Cell-Free (In Vitro) Protein Expression How It Helps Speed Up Your Research Gary Kobs January 14, 2014 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 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 • • Combined Usage of HaloTag ® and Cell-Free Expressed Proteins • • A unique, multi-functional protein fusion tag for global protein characterization Protein kinase assays and inhibitor screen Summary Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 2 Easiest/Fastest Method from DNA to Protein Saves valuable time • Produce protein in 1-2 hours vs. days to Cell-free competent plasmid or weeks in E. coli or mammalian cells - None Template Label - 35S-methionine - Biotinylated lysines - Fluor-labeled lysines Cell-free competent PCR product TNT® System Master Mix • • • • • • Use PCR product templates and bypass cloning ORF into expression vector Produces sufficient protein for many applications including: • Protein:protein interactions T7 RNA polymerase Cellular lysate Nucleotides Amino acids Energy regenerating system 1-2 hr @ 37°C Transcription/translation • 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 Protein of Interest Simplifies detection • Directly label protein during synthesis Multiple Applications Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. • Fluorophore, 35S, biotin 3 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 Most active protein Native mammalian system Highest yield Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. Maximal soluble protein 4 Faster Protein Production than E. coli Systems E. coli Expression Vector Prep Template Prep TNT® System Analysis or Purification Analysis or Purification 2 hrs 2 days Critical Timeline Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 5 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 10 100 500 Protein Yield (µg/ml) Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 6 Characteristics of Cell-Free Expression Systems System Time Yield TNT® T7 or SP6 Quick Coupled System (RRL) 1 hour 0.5µg/50µl Any vector containing a T7 or SP6 promoter upstream of coding sequence. S30 High Yield (Bacterial) 1 hour 25µg/50µl 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). TNT® SP6 High-Yield Wheat Germ System 2 hours 5-12.5µg/50µl TNT® T7 Insect Cell System 4 hours 4.0µg/50µl Optimized Wheat Germ Vector Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. Requirements Require use of a baculovirus expression vector with T7 promoter upstream. Insect Cell Vector Example 7 Fluorescent Detection of Expressed Proteins Non-Radioactive Co-translational Labeling TNT® T7 Quick Coupled System Proteins expressed TNTT7 Quick Coupled system 50kDa 50kDa 37kDa 38 kDa 37kDa 24 kDa 25kDa 25kDa 17 kDa 4 Proteins 5 Lysate Only Lysate only FluoroTect™ Green 3 Protein 5 lys 2 Protein 4 1 lys Protein 1 lys 52 kDa Protein 3 Translation reaction + FluoroTect™ GreenLys tRNA 250kDa 150kDa 100kDa 75kDa 250kDa 102 kDa 150kDa 100kDa 76 kDa 75kDa Protein 2 FluoroTect™ GreenLys tRNA TNT S 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… Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 8 Indirect Detection of In Vitro Expressed Proteins Transcend™ Biotin Co-translational Labeling Proteins expressed TNTT7 Quick Coupled system TNT® SP6 Wheat Germ High-Yield System Proteins expressed TNT SP6 High Yield Wheat Germ system 102 kDa 102 kDa 102 kDa Transcend™ 7676 kDa kDa kDa 5252 kDa Biotinylated tRNA 76 kDa 52 kDa 3838 kDa kDa 1 2 3 4 Proteins 5 Lysate Only Lysate only Lysate only Protein 5 Protein 4 Biotin Protein 3 lys Protein 2 lys Protein 1 lys Protein 5 1717 kDa kDa 17 kDa Protein 4 tRNA Protein 3 2424 kDa kDa Protein 2 Translation reaction + 24 kDa Transcend™ Biotinylated Protein 1 38 kDa 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… Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 9 Detection Sensitivity Rivaling 35S-Methionine Fluorescent 5µl of reaction Direct in-gel detection Biotin, Indirect 1µl of reaction Chemiluminescent Detection 20µCi 35S-Met 1µl of reaction overnight exposure Radioactive 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, 23-7. Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 10 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 + Label Lysine 25%-33% of lysines get labeled Precharged Transcend™ or FluoroTect™ tRNA UUU UUU AAA/G 5’ 3’ Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 11 E. coli S30 Cell-Free Systems High Yield System Amenable to HTS Applications Key Advantages Bacteria • 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 S30 T7 High-Yield Protein Expression System Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. • Well suited to protein labeling and large-scale production • Documented success in membrane protein production General Vector/Template Requirements • T7 vectors for E. coli cell-based expression • Specialized vectors available 12 E. coli S30 Cell-Free Systems Protein Expression/Purification Manual MagneHis™ Purification Fluorescent Detection Cell-Free E. coli Expression pFN6A-mgGFP Clone HQ Monster Green® GFP S30 T7 High Yield System Automated 1-16 Sample Purification (Maxwell® 16) Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 13 Wheat Germ Cell-Free Systems More Soluble Protein from an Animal-Free System Key Advantages Plant • 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. • SP6 or T7 promoter vectors depending on WG kit • Specialized vectors with plant viral 5’, 3’ UTRs for higher expression available 14 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 15 Insect Cell-Free Systems Highly Active Protein from an Animal-Free System Key Advantages Insect • Production of highly active, full-length proteins • Relatively high protein yields • 75µg/ml in TNT® coupled system • Animal-free, eukaryotic system General Vector/Template Requirements TNT® T7 Insect Cell Extract System Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. • T7 promoter vectors • Specialized vectors available with baculovirus 5’, 3’ UTRs for higher expression 16 Insect Cell-Free Systems Highly Active Protein from an Animal-Free System Highest Specific Activity cPKA with T NT® T7 Insect Cell System 35 Protein Yield (WG>ICE>RR) 30 Tested: ng/ul 25 TNT® SP6 High Yield Wheat Germ System TNT® T7 Quick (Rabbit Retic.) System TNT® T7 Insect Cell Extract System 20 15 10 5 Fold (specific activiy/TNTR) 0 5 4 3 WG RR ICE no cAMP cAMP Specific Activity (ICE>RRWG) 2 1 0 WG Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. RR ICE 17 Rabbit Retic (Mammalian) Cell-Free Systems Mammalian Protein Expression in a Native Setting Key Advantages Mammalian • Native system for mammalian proteins • Post-translational processing • Adding canine microsomal membranes • Full-length protein expression likely General Vector/Template Requirements TNT® Quick Coupled T7 and SP6 Systems • T7 or SP6 promoter vectors • T7 TNT® System: Compatible with many templates • Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. A “Universal Lysate” 18 Rabbit Retic (Mammalian) Cell-Free Systems “Universal Lysate” Suitable for Many T7 Vectors Compatibility with Multiple T7 Expression Vectors T7 Vector 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 19 Rabbit Retic (Mammalian) Cell-Free Systems Rapid Protein Expression/Functional Screening Easily Test Expression Vectors Containing T7 Promoters w/TNT® T7 Quick pET15b pF1A pF4A His Renilla luciferase T7t lacO rbs Renilla luciferase T7t Intron T7p lacO T7p T7p CMVp rbs Renilla Expression Renilla luciferase SV40 Late PolyA Signal Renilla Luciferase Activity 16000 14000 12000 RLU 10000 8000 6000 4000 2000 0 control Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. pET15b pF1A pF4A 20 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 35S-Met Western blotting detection using primary antibody to the expressed protein Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 21 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 22 Protein:Protein Interactions: Testing for Direct Interaction of Asr1 with the RNAP II CTD Daulny A., et al. (2008) Proc. Nat. Acad. Sci. 105, 19649-54 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 23 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 PO4 +/- Phosphorylation E. coli SDS-PAGE & transfer to membrane Incubate Wash Detect bound Asr1 by autoradiography 35S 1 hour + 35S-Met Asr1 TNT® T7 Quick Coupled System Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 24 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, 19649-54 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 25 Protein:DNA Binding and Protein:Protein Interaction Analysis of AHRR1 Protein Evans B., et al. (2008) Mol. Pharm. 73, 387-98 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 26 Protein:DNA Binding: Testing the DNA Binding Activity of AHHR1 and ARNT2 Experimental Design - Gel Shift Assays using Cell-Free Expressed Proteins 1 hour unlabeled AHRR1 Wild Type Y9F Mutant TNT® T7 Quick Coupled System 32P Mix & Incubate 1 hour unlabeled Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. ARNT2 AHRR1 ARNT2 32P Run in a nondenaturing polyacrylamide gel & autoradiograph 27 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, 387-98 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 28 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 • 35S-met labeled AHRR1 proteins • Expressed ARNT2 protein using same system – unlabeled AHHR1 FL & Deletions + 35S-Met ARNT2 • Used anti-ARNT2 antibody in CoIP protein complexes • Detected Co-IP’d AHRR1 proteins by SDS-gel & fluorography Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 29 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 189-270 are required for the interaction Evans B., et al. (2008) Mol. Pharm. 73, 387-98 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 30 In Vitro Functional Analysis of the CrPV IRES Garrey J., et al. (2010) J. Virol. 84, 1124-38 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 31 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 30 min +35S-Met R luc 4E-BP1 FF luc Dual-Luciferase Assays R Luc + FF Luc 0, 30, 60 min time points TNT® T7 Quick Coupled System SDS gel followed by autoradiography Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 32 In Vitro Functional Analysis of the CrPV IRES Results In Vitro Translation Data 1. Both IRES’s promote translation of FF luc in the second cistron 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, 1124-38 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 33 Generation of Labeled Proteins for Structural Analysis by Solution NMR Zhao, Li et al. (2010) Struc.Funct.Gen.11, 201-209 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 34 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 35 Ubiquitination Analysis of p73 Suppressor Protein Jung, Y-S et al. (2011) J. Biol. Chem. 286, 35388-95 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 36 Use of Cell-Free for Ubiquitination Analysis Results 1. 1PirH2 physically interacts with p73 2. Pirh2 promotes p73 polyubiquitination Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 37 Membrane Association of Pestivirus Glycoprotein Erns Tews, B-A. et al. (2007) J. Biol. Chem. 282, 32730-41 Key Experiments Using Cell-free Expressed Proteins • In vitro translation of wild type Erns and Erns containing mutations • Proteinase K protection assays in the presence of canine microsomal membranes Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 38 Membrane Association of Pestivirus Glycoprotein Erns Results 1. Proteinase K protection assays showed show that Erns 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 39 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 40 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 41 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 42 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 HaloTag® Protein of Interest + N- or C- terminal fusions Chloroalkane Linker O Cl O Functional Group Binding and… • 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, 110-120. Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. Protein of Interest O O Functional Group Covalent bond between HaloTag® and chloroalkane (+ functional group) 43 Many Functional Groups are Available to Match Your Research Application(s) Protein of Interest O Functional Group O O Cl O HaloTag® Surfaces/Resins Capture and Display Protein arrays Purification Interaction analysis O Cl O Cl Cl Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. HaloTag® Fluorescent Ligands Labeling and Detection O Cellular imaging Gel analysis Quantitation O O HaloTag® Reactive Ligands Custom Modifications O Attach to particles, surfaces Attach special ligands 44 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 Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 45 Enzymatic Assays Measuring Protein Kinase A (PKA) Activity Experimental Design – Immobilization of HaloTag®-cPKA followed by Kinase Assays Immobilized Enzyme HT-cPKA Expression Plasmid HaloLink® Magnetic Beads Cl O cPKA O Kinase Assay Measure Remaining ATP Kinase-Glo® O Incubate O Purify cPKA PKA Activity ADP ATP HaloTag® TNT® SP6 High Yield Wheat Germ System HaloLink™ Array Slide PKA Kinase Assay OO ©2013 Promega Corporation. Proprietary Information. Not for further distribution. OO OO Promega Corporation ProFluor® PKA Activity 46 Enzymatic Assays: Testing for PKA Inhibitors +/- Inhibitors O O WGE ICE Compound U0126 H89 ProFluor® Kinase Assay Higher Bars = More Inhibition Target MEK1/2 PKA specific cPKA Inhibition No Yes PKI PKA specific Yes Staurosporine Non-selective Kinase inhibitor Yes DMSO No 100 20 ul particles 10 ul particles 80 0.3 u rPKA 0.075 u rPKA 60 40 WGE 20 0 U0126 Percent Maximum Signal (Inhibition) cPKA PKA Inhibitor Results Percent Maximum Signal (Inhibition) PKA Inhibitor Screening Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. PKI st aurosporine DMSO Buf f er Inhibitor (10 µM) 20 ul particles 80 10 ul particles 0.3 u rPKA 60 0.075 u rPKA 40 ICE 20 0 U0126 Donna M. Leippe, Kate Qin Zhao, Kevin Hsiao and Michael R. Slater. Analytical Chemistry Insights (2010)5, 25-36 H89 100 H89 PKI st aurosporine DMSO Buf f er Inhibitor (10 µM) 47 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… Promega Corporation ©2013 Promega Corporation. Proprietary Information. Not for further distribution. 48
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