Table Of A. PopCulture Quick Screen For Expression Level .

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Novagen pET System ManualTable ofContentsI. 11th EditionAbout the System3A. DescriptionTABLE OF CONTENTS3A. PopCulture Quick Screen forExpression Level, Activity,and Solubility25B. Licensing and Use Agreement3B. Total Cell Protein (TCP) Fraction25C. System Components4C. Medium Fraction255D. Periplasmic Fraction26E. Soluble Cytoplasmic Fraction28II. Getting StartedA. Overview of thepET System Process5B. Growth Media and Antibiotics6BugBuster Master Mixtreatment2829Growth media6Antibiotics and stock solutions7rLysozyme Solution andfreeze/thaw treatmentC. Vector Preparation7Mechanical disruption29D. Insert Preparation9F. Insoluble CytoplasmicFraction31III. Cloning Inserts in pET Vectors10A. Ligation10B. Transformation10Handling tips11Procedure12Plating techniques13ColiRollers Plating Beads14Standard spreader14C. Storage of Strains14D. Analysis of pET RecombinantsInclusion body purificationafter BugBuster Master Mixtreatment31Inclusion body purificationafter mechanical cell lysisor rLysozyme treatment32G. Preparation of Extracts withPopCulture Reagent32H. Detecting and QuantifyingTarget Proteins3415Normalized SDS-PAGEgel loading35Ligation screening15VI. Purifying Target Proteins36Colony screening16A. Protein Purification36Plasmid preparation procedure18B. Solubilization andRefolding Proteins36VII. Additional Guidelines37IV. Expressing the Target Gene19A. Bacteriophage CE619B. Expression Host Transformation 19C. Induction of λDE3 Lysogenswith IPTG19Preparation for induction19Sample induction protocol20D. Autoinduction of λDE3 Lysogens 20Overnight Express Instant TB MediumV. Target Protein Verification2124A. Choosing a pET Vector37Solubility and cellularlocalization38Fusion tags39Antibiotic resistanceB. pET Vector Cloning Strategies4043Produce recombinant proteinswithout fusions43Produce recombinantproteins without fusionsafter protease cleavage44Normalizing loading volumesfor SDS-PAGE24Ligation-independent cloning44Optical density analysis of theinduced culture24C. Regulating Protein Expressionin the pET System45TB055 11th Edition 01/06USA and CanadaTel 800 526 7319novatech@novagen.comUnited Kingdom and IrelandUK Freephone 0800 622935Ireland Toll Free 1800 ephone 0800 100 3496techservice@merckbiosciences.deAll Other CountriesContact Your Local Distributorwww.novagen.comnovatech@novagen.com1

Novagen pET System ManualTABLE OF CONTENTS 11th EditionThe T7lac promoter45Lysis buffer56Transcription terminator65pLysS and pLysE hosts45Periplasmic localization57Cytoplasmic localization58Instability of the target mRNAand protein65Host strains58Vector and hostcombinations affectexpression levels46Media containing glucose47pLacI hosts47pETcoco SystemIX. Acknowledgements65C. Correcting forRare Codon UsageX. References6659XI. Index6948D. Toxic Genes andPlasmid Instability60XII. Academic & Non-profitLaboratory Assurance Letter71D. Hosts for Cloning48Use of ampicillin60E. Hosts for Expression49Supplementing with glucose61XIII. Bacterial Strain Non-DistributionAgreement7250Stabilize a toxic gene in anampR pET vector forglycerol stock storageXIV. Appendix:73Protease deficiencyAdjustable expressionlevels throughout allcells in a culture50Disulfide bond formationand solubility enhancement 50Rare tRNA supplementation 51Selenomethionine labelingpET system host straincharacteristics table5152E. Coexpression oftarget proteins61λDE3 lysogens forprotein expressionCoexpression vectors andadaptors62Replicons, drug resistance,and copy numberIsogenic host strains for initialcloning, controls,and expression7563F. Other Factors InfluencingExpression6355N-end rule63A. Induction Controls5556Secondary site translationinitiation6456Secondary structure in themRNA transcript6456Unexpected stop codons65B. Enhancing Solubilityand FoldingTemperature7362VIII. Optimizing Expressionβ-galactosidase assayA. pET System Host Strains andLambda Phage73pET host strain competentcell sets76pET System lambda phage76B. Detection/Assay Toolsfor Fusion Tag Proteins77C. Purification Tools78 2005 EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt, Germany.The Novagen name, Novagen logo, BugBuster , His Bind , His Tag , HSV Tag , NovaTope , Pellet Paint , PopCulture , and T7 Tag are registered trademarks ofEMD Biosciences, Inc. in the United States and certain other jurisdictions. Beta Red , Clonables , ColiRollers , DsbA Tag , DsbC Tag , Ekapture , FRETWorks , GST Bind ,GST Mag , GST Tag , His Mag , LIC Duet , LumiBlot , Lysonase , Mobius , NovaTaq , Nus Tag , OnEx , Overnight Express , Perfect DNA , Perfect Protein , pETBlue ,pETcoco , RoboPop , Rosetta , RosettaBlue , Rosetta-gami , S Tag , Singles , SpinPrep , Strandase , Trail Mix , TriEx , Trx Tag , Tuner , and Xarrest are trademarksof EMD Biosciences, Inc. Fractogel and Benzonase are registered trademarks of Merck KGaA, Darmstadt, Germany. MagPrep is a registered trademark of EMD Chemical, Inc.Sonifier is a registered trademark of Branson Instruments, Inc. MacroPrep is a registered trademark of Bio-Rad Laboratories, GmbH, Ltd. Cy5 is a trademark of GE Healthcare. Strep Tactin and Strep Tag are registered trademarks of IBA GmbH. PicoGreen is a registered trademark of Molecular Probes, Inc. Triton is a registered trademarkof Rohm and Haas Co. Superflow is a trademark of Sterogene Bioseparations Inc. SpeedVac is a registered trademark of Thermo Savant, Inc. BugStopper is a trademark ofWhatman International, Ltd.The pET expression system is covered under U.S. Patent Nos. 4,952,496; 5,693,489; and 5,869,320. For academic and non-profit laboratories, a nondistribution agreementaccompanies the products. Commercial laboratories must obtain a research-use license from Brookhaven Science Associates prior to purchase of the products.The pET-32 and pET-48 vectors are sold under patent license from Genetics Institute, Inc. For research use only. Licenses for commercial manufacture or use may be obtaineddirectly from Genetics Institute Inc., 87 Cambridge Park Drive, Cambridge, MA 02140. Vectors containing the His Tag sequence are licensed under U.S. Patent Nos. 5,310,663;5,284,933; and European Patent No. 282,042 issued to Hoffmann-La Roche, Inc., Nutley NJ and/or Hoffmann, La Roche, Ltd. Basel, Switerzerland and are provided only for usein research. Information about license for commercial use is available from QIAGEN GmbH, Max-Volmer-Str. 4, D-40724 Hilden, Germany. Ni-NTA His Bind Resins are manufactured by QIAGEN GmbH. His Tag Monoclonal Antibody is covered under German Patent, No. DE 19 507 166 and international patent applications are filed under W09626963and is provided only for use in research. Information about license for commercial use is available from QIAGEN GmbH, Max-Volumer-Str. 4, D-40724 Hilden, Germany. TheGST Tag technology is covered under U.S. Patent No. 5,654,176, European Patent No. 293,249B1, and Australian patent No. 607,511. Vectors containing a Strep Tag sequencefor purification and detection are covered under U.S. Patent No. 5,506,121, U.K. Patent No. 2,272,698, and French Patent No. 93 13 066; Strep Tactin is covered by U.S. PatentNo. 6,103,493 issued to IBA GmbH. Strep Tactin resins, Strep Tactin buffers, and Strep Tag antibodies are manufactured by IBA GmbH. These products are provided only for usein research. Information about commercial use is available from IBA GmbH, Rudolf-Wissell-Str. 28, D-37079 Goettingen, Germany.NovaTaq and KOD DNA Polymerase are sold under licensing arrangements with F. Hoffmann La-Roche Ltd., and Roche Molecular Systems, Inc. KOD DNA Polymerases are manufactured by Toyobo and distributed by EMD Biosciences, Inc., Novagen brand. KOD products are not available through Novagen in Japan. KOD XL DNA Polymerase is licensedunder U.S. Patent No. 5,436,149 owned by Takara Shuzo, Co., Ltd. Purchase of KOD or NovaTaq DNA Polymerases is accompanied by a limited license to use it in the PolymeraseChain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either bypayment to Applied Biosystems or as purchased, i.e., an authorized thermal cycler.2USA and CanadaTel 800 526 7319novatech@novagen.comUnited Kingdom and IrelandUK Freephone 0800 622935Ireland Toll Free 1800 ephone 0800 100 3496techservice@merckbiosciences.deAll Other CountriesContact Your Local Distributorwww.novagen.comnovatech@novagen.com

Novagen pET System ManualI.About theSystem 11th EditionIABOUT THE SYSTEMA. DescriptionThe pET System is the most powerful system yet developed for the cloning and expressionof recombinant proteins in E. coli. Target genes are cloned in pET plasmids undercontrol of strong bacteriophage T7 transcription and (optionally) translation signals;expression is induced by providing a source of T7 RNA polymerase in the host cell. T7RNA polymerase is so selective and active that, when fully induced, almost all of thecell’s resources are converted to target gene expression; the desired product can comprisemore than 50% of the total cell protein a few hours after induction. Although this systemis extremely powerful, it is also possible to attenuate the expression level simply bylowering the concentration of inducer. Decreasing the expression level may enhancethe soluble yield of some target proteins. Another important benefit of this system is itsability to maintain target genes transcriptionally silent in the uninduced state. Targetgenes are initially cloned using hosts that do not contain the T7 RNA polymerase gene,thus eliminating plasmid instability due to the production of proteins potentially toxicto the host cell. Once established in a non-expression host, target protein expressionmay be initiated either by infecting the host with λCE6, a phage that carries the T7RNA polymerase gene under the control of the λ pL and pI promoters, or by transferringthe plasmid into an expression host containing a chromosomal copy of the T7 RNApolymerase gene under lacUV5 control. In the second case, expression is induced by theaddition of IPTG or lactose to the bacterial culture or using an autoinduction medium.Although in some cases (e.g., with innocuous target proteins) it may be possible to clonedirectly into expression hosts, this approach is not recommended as a general strategy.Two types of T7 promoters and several hosts that differ in their stringency of suppressingbasal expression levels are available, providing great flexibility and the ability tooptimize the expression of a wide variety of target genes.All of the pET vectors and companion products are available as kits designed forconvenient cloning, expression, detection, and purification of target proteins. The pETExpression Systems provide the plasmids and host strains. The background informationin Section VII, Additional Guidelines, will help you determine the best vector/hostcombination for your application.B. Licensing and Use AgreementThis T7 expression system, including bacteria, phages, and plasmids that carry the genefor T7 RNA polymerase, is made available under the conditions listed in the Academicand Non-profit Laboratory Assurance Letter. Please refer to the complete list ofconditions on page 71.USA and CanadaTel 800 526 7319novatech@novagen.comUnited Kingdom and IrelandUK Freephone 0800 622935Ireland Toll Free 1800 ephone 0800 100 3496techservice@merckbiosciences.deAll Other CountriesContact Your Local Distributorwww.novagen.comnovatech@novagen.com3

INovagen pET System ManualABOUT THE SYSTEM 11th EditionC. System ComponentspET Expression Systems provide the core reagents needed for target gene cloning andexpression. pET vector DNA, 10 µg each of the indicated plasmids Host bacterial strains BL21, BL21(DE3), and BL21(DE3)pLysS, glycerol stocks1, 2A good way to distinguishglycerol stocks from competent cells: glycerol stocks aresupplied in screw-top tubes;competent cells have a fliptop. Induction control clone, glycerol stockSystems plus Competent Cells include all of the above listed components and aset of three competent host strains ready for high-efficiency transformation of pETrecombinants. The competent cells are sufficient for up to 10 transformations in eachhost: 0.2 ml each of NovaBlue, BL21(DE3), and BL21(DE3)pLysS Competent Cells1, 2 SOC medium Test Plasmid1The pET Peptide Expression System 31 includes host strains BLR and BLR(DE3)pLysS in place ofthe BL21 series hosts.2The pET Trx Fusion System 32 includes the Origami series hosts strains in addition to the BL21 series hosts.Separate components and related products: see the Novagen catalog or Novagen website (www.novagen.com) for a complete listing of pET vectors, systems, and competentcells.4USA and CanadaTel 800 526 7319novatech@novagen.comUnited Kingdom and IrelandUK Freephone 0800 622935Ireland Toll Free 1800 ephone 0800 100 3496techservice@merckbiosciences.deAll Other CountriesContact Your Local Distributorwww.novagen.comnovatech@novagen.com

Novagen pET System ManualII.GettingStarted 11th EditionIIGETTING STARTEDA. Overview of the pET System ProcessChoose a pET Vector (page 37) Application of the expressed protein Specific information known about target protein Cloning strategySolubility and cellular localizationsFusion tags; need for tag removalRegulation of protein expression, (i.e.,promoter and expression strain)Prepare Insert DNA (page 9)Prepare pET Vector (page 7) Plasmid and/or PCR DNA Restriction digest or generateLIC overhangs Gel-purify Digest with restriction enzyme(s) anddephosphorylate (or use LIC vector) Gel-purify (or use LIC vector)Clone Insert into pET Vector (page 10) Ligate or anneal insert with pET vector Transform into non-expression host (e.g.,NovaBlue) Identify positive clones; colony PCR,prep

Novagen pET System Manual 11th Edition TABLE OF CONTENTS TB055 11th Edition 01/06. 2 USA and Canada United Kingdom and Ireland Germany All Other Countries Tel 800 526 7319 UK Freephone 0800 622935 Freephone 0800 100 3496 Contact Your Local Distributor novatech@novagen.com Ireland Toll Free 1800 409445 techservice@merckbiosciences.de www.novagen.com techservice@merckbiosciences.co.uk .

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