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pET SYSTEM

pET SYSTEM

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Published by navyeinstein1991

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Published by: navyeinstein1991 on Apr 16, 2011
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08/20/2011

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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 Distributornovatech@novagen.com Ireland Toll Free 1800 409445 techservice@merckbiosciences.de www.novagen.comtechservice@merckbiosciences.co.uk novatech@novagen.com
Table of Contents
 
TABLE OF CONTENTS
Novagen
 
 
  pET System Manual  
 
11th Edition
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 Distributornovatech@novagen.com Ireland Toll Free 1800 409445 techservice@merckbiosciences.de www.novagen.comtechservice@merckbiosciences.co.uk novatech@novagen.com
Novagen
 
 
  pET System Manual  
 
11th Edition
© 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 of EMD 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™, Nova
Taq
™, 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 Health-care. 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 of Whatman 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 manufac-tured 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.Nova
Taq
™ and KOD DNA Polymerase are sold under licensing arrangements with F. Hoffmann La-Roche Ltd., and Roche Molecular Systems, Inc. KOD DNA Polymerases are man-ufactured 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 Nova
Taq
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.
TABLE OF CONTENTS
β
-
R
 
Novagen
 
 
  pET System Manual  
 
11th Edition
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 Distributornovatech@novagen.com Ireland Toll Free 1800 409445 techservice@merckbiosciences.de www.novagen.comtechservice@merckbiosciences.co.uk novatech@novagen.com
I ABOUT THE SYSTEM
A. Description
The 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 under control 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
λ
CE6, a phage that carries the T7RNA polymerase gene under the control of the
λ
 p
L
and
 p
I
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 for convenient 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 Agreement
This 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 of conditions on page 71.
I.About theSystem
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