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IV. Expressing the Target Gene
A. Expression Host Transformation B. Induction of λDE3 Lysogens Preparation for Induction Sample Induction Protocol C. Optimizing Expression Plasmid Stability Test D. Solubility Formation of Disulfide Bonds E. Toxic Genes and Plasmid Instability
38Байду номын сангаас
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VII. Induction Control: β -Galactosidase Recombinant
β -Galactosidase Assay
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VIII. Acknowledgments IX. References X. Index XI. Academic and Non-profit Laboratory Assurance Letter XII. AD494, BL21trxB, Origami and Origami B Non-Distribution Agreement XIII. Related Products and Separate Components
pET System Manual
Table of Contents
Table of Contents I. About the System
A. B. C. D. Description Licensing and Use Agreement System Components The pET Vectors Vector Characteristics and Cloning Strategy Ligation-Independent Cloning (LIC) of PCR Products Fusion Tags Antibiotic Resistance pET Vector Characteristics Hosts for Cloning Hosts for Expression pET System Host Strain Characteristics Selecting Host Strains List of pET System Host Strains and Lambda Phages Media Containing Glucose The T7lac Promoter pLysS and pLysE Hosts pLacI Hosts Bacteriophage CE6 Induction Controls
United States & Canada 800-207-0144 Germany 0800 6931 000 United Kingdom 0800 622935 Or your local sales office
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TB055 9th Edition 05/00
V.
Detecting and Quantifying Target Proteins
Detection/Assay Products for Fusion Tags
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VI. Purifying Target Proteins
A. Small Scale Analysis Growth and Induction Optical Density Analysis of the Induced Culture Total Cell Protein (TCP) Sample Media Sample Periplasmic Fraction Sample–Osmotic Shock Soluble Cytoplasmic Fraction Insoluble Cytoplasmic Fraction ® B. Preparation of Extracts with BugBuster™ Reagent and Benzonase Nuclease BugBuster/Benzonase Soluble Fraction BugBuster/Benzonase Inclusion Body Purification C. SDS-PAGE and Western Blot Analysis Normalized SDS-PAGE Gel Loading D. Large Scale Induction and Fractionation Media Fraction Periplasmic Fraction Soluble Whole Cell Extract Fraction Insoluble Whole Cell Extract Fraction–Isolation of Inclusion Bodies Solubilization of Inclusion Bodies and Refolding Proteins
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E. F. G. H. I. J. K. L. M. N. O.
II.
Getting Started
A. B. C. D. The pET System Process Growth Media Storage of Strains Vector Preparation Recommendations E. Insert Preparation
Novagen
1
pET System Manual
Use of Ampicillin Precautions to Maximize Expression Rationale for Plasmid Stability Test F. Difficult Target Proteins Other Factors Influencing Expression Level 33 33 34 34 36
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Copyright ã 1992–2000 by Novagen, Inc. All rights reserved. BugBuster, CBD•Tag, Clonables, Dsb•Tag, EcoPro, EKapture, EXlox, FRETWorks, GST•Bind, GST•Tag, His•Bind, His•Tag, HSV•Tag, LumiBlot, Mobius, NovaTope, Nus•Tag, Origami, Pellet Paint, Perfect DNA, Perfect Protein, pETBlue, pSCREEN, S•Tag, Single Tube Protein, Singles, SpinPrep, STP3, Strandase, T7 •Tag, TriEx, Trx•Tag, Tuner, UltraMobius, Xarrest, the Novagen name and logo are trademarks and registered trademarks of Novagen, Inc. Benzonase is a trademark of Benzon Pharma A/S. Superflow is a trademark of Sterogene Bioseparations Inc. Fractogel is a trademark of Merck KGaA, Darmstadt, Germany. MagPrep is a trademark of EM Industries Inc. CBIND is a trademark of CBD Technologies, Inc. Novagen’s primers are designed and sold for use in the Polymerase Chain Reaction (PCR) process covered by patents owned by Hoffmann-La Roche. Use of the PCR process requires a license. A license for research may be obtained by purchase and use of authorized reagents and DNA thermal cyclers from the Perkin-Elmer Corporation or by otherwise negotiating a license with Perkin-Elmer. Triton is a trademark of Rohm and Haas Co. The pET system is covered by U.S. Patent no. 4,952,496. A non-distribution agreement accompanies the products. Commercial customers must obtain a license agreement from Brookhaven Science Associates before purchase. The pET-32 vectors are sold under patent license from Genetics Institute, Inc. For research use only. Licenses for commercial manufacture or use may be obtained directly from Genetics Institute, Inc., 87 Cambridge Park Drive, Cambridge, MA 02140. The CBD•Tag technology is covered under U.S. Patent nos. 5,496,934; 5,202,247; 5,340,731; and 5,137,819. Use of this technology for commercial purposes requires a license from CBD Technologies, Inc. Ni-NTA His•Bind Resins are manufactured by Qiagen GmbH. His•Tag Monoclonal Antibody is covered under German Patent No. DE 19507 166. The GST•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 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, Switzerland and are provided only for use in research. Information about licenses for commercial use is available from QIAGEN GmbH, Max-Volmer-Str. 4, D–40724 Hilden, Germany.
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III. Cloning Inserts in pET Vectors
A. Ligation B. Transformation Handling Tips Procedure Plating Technique C. Analysis of pET Recombinants ® Transcription/Translation Analysis with EcoPro™ or STP3 Systems Plasmid Templates PCR Templates Ligation PCR for Transcription/Translation Analysis Colony PCR for Transcription/Translation Analysis Colony Screening Plasmid Miniprep Procedure Sequencing
