Choose the convenient way to move from genes to functional proteins. Generate Some Time to. focus on what matters. Gene Fusion System AB

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1 Choose the convenient way to move from genes to functional proteins Generate Some Time to focus on what matters AB ST Gene Fusion System

2 In proteomics, speed and accuracy matter Spend some time to save time - can you afford not to read on? Dr. Darcy Birse Department of Biochemistry and Biophysics, Stockholm University. Amersham Biosciences GST products provide scientists with the freedom to do experiments from gene to function to structure in one complementary system. GST Gene Fusion System keeps you on the fast track When working with fusion proteins, essential factors include purity, speed, yield, preservation of protein structure, and experimental reproducibility. Glutathione S-Transferase (GST) Gene Fusion System provides solutions to these factors through an integrated system for the expression, purification and detection of fusion proteins produced in E. coli. GST Gene Fusion System consists of three key components: pgex plasmid vectors, GST Purification Modules, and GST Detection Modules. Companion products such as site-specific proteases, purification products and instruments complement the range. There are many reasons to choose the GST Gene Fusion System, from faster methodology to higher yields and purity of functional proteins. 2

3 Speed counts Research today is competitive and in the race for results in proteomics, a fast methodology that delivers reliable results is always advantageous. One-step GST tag removal and efficient protein purification will save you both time and effort. Protein friendly What is the point of spending weeks producing fusion proteins if the purification process damages them? pgex-6p PreScission Protease vectors offer GST tag removal and protein purification at 4 C for improved protein stability. Increased yield and purity A minimal number of steps and highly specific affinity purification mean higher yields and purity. Single source for GST expertise Amersham Biosciences offers a complete solution for working with GST fusion proteins. From everything you need for success on a lab scale to large-scale purification, Amersham Biosciences offers the reagents, equipment, and expertise designed to meet your needs - see for details. 2

4 Expression Good research starts at the beginning pgex vectors ensure high yields of fusion proteins GST Gene Fusion System incorporates pgex plasmids for inducible, high-level intracellular expression of genes or gene-fragments as fusions with Schistosoma japonicum GST. Expression in E. coli yields fusion proteins in the cytoplasm with the GST moiety at the amino terminus and the protein of interest at the carboxyl terminus. Ten pgex vectors are available in three series - make your choice based on reading frame and the protease that you want to use downstream. 4

5 pgex fusion vectors offer: A tac promoter for chemically-inducible, high-level expression. An internal laciq gene so you can use any E. coli host. A lyophilized culture of E. coli BL21 is supplied with each vector. This strain is deficient in OmpT and Lon proteases, which minimizes proteolytic degradation of fusion proteins. Choice of protease recognition sites for cleaving the target protein from the fusion product. pgex-6p PreScission Protease vectors offer the most efficient method for cleaving and purifying GST fusion proteins. Site-specific cleavage is performed with simultaneous immobilization of the protease on the column. The protease and GST tag are removed in a single step. Other vectors available include pgex-5x Factor Xa vectors and pgex-4t Thrombin vectors, as well as pgex-2tk, which allows the detection of expressed proteins by direct labelling of fusion products in vitro. For detailed methodologies, see our technical handbooks listed on page 13. 5

6 Purification Why settle for less than 90% purity? Choose the purification format that fits your scale GST purification features Glutathione Sepharose affinity media for rapid, mild affinity purification of GST fusion proteins. Fusion proteins can be purified to greater than 90% in a single step using mild, nondenaturing elution conditions with 10 mm reduced glutathione at ph 8.0 that preserves protein antigenicity and function. You can easily scale up from microgram to hundreds of milligram yields. MicroSpin GST Purification Module MicroSpin GST Purification Module comprises 50 prepacked Glutathione Sepharose 4B columns in an ideal microcentrifuge format. Each column, with its 50 µl bed volume, is sufficient to purify up to 400 µg of recombinant GST. GSTrap FF GSTrap FF columns are prepacked 1 ml and 5 ml HiTrap columns containing Glutathione Sepharose 4 Fast Flow with a binding capacity of mg GST/ml medium. GSTrap FF columns are easy-to-use with either a simple syringe, a laboratory pump or a chromatography system, such as ÄKTAprime. 6

7 GSTPrep FF 16/10 GSTPrep FF 16/10 columns are prepacked 20 ml HiPrep columns containing Glutathione Sepharose 4 Fast Flow for preparative purifications of GST fusion proteins. Each column binds more than 200 mg fusion protein. Glutathione Sepharose 4 Fast Flow Glutathione Sepharose 4 Fast Flow is supplied as pre-swollen bulk medium, and is an excellent choice for column purification. Glutathione Sepharose 4B Glutathione Sepharose 4B is supplied as pre-swollen bulk medium, and is an excellent choice for batch purifications. 7

8 Tag removal 8 On-column cleavage with PreScission Protease Why not purify the target protein at the same time as removing the GST tag? On-column cleavage is generally recommended as the method of choice since many contaminants can be washed out and the cleaved target protein eluted with a higher level of purity. With the pgex-6p vectors, you can use PreScission Protease to cleave the GST tag from the fusion protein. PreScission Protease is a fusion protein of GST and human rhinovirus 3C protease. PreScission Protease works best at 4 C, unlike other proteases that requiretemperatures closer to 20 C. A lower temperature means increased protein stability. The amino acid sequence recognized by PreScission Protease is synthetic. This means that the enzyme will not cleave your target protein. Unlike other proteases, once the GST tag is cleaved off, only a glycine-proline group remains at the amino terminus. This group has virtually no functional or conformational effect on your protein (the actual number of non-native amino acids at the amino terminus will depend on which restriction site was used for cloning). Only the target protein is eluted from the column while bound to Glutathione Sepharose. GST and PreScission Protease remain bound to the media. You can expect purity levels in excess of 90%. For detailed methodologies, see our technical handbooks listed on page 13.

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10 Detection Spend less time looking GST 96-Well Detection Module ready-coated for high-throughput screening GST 96-Well Detection Module permits rapid, sensitive determination of GST fusion proteins in a variety of samples. Clarified lysates or intermediate purification fractions can be applied directly into the wells of GST 96-Well Detection Plates. GST fusion proteins are captured by specific binding to anti-gst antibody that is immobilized on the walls of each well. 10

11 Detect captured fusion proteins with the provided horseradish peroxidase conjugated anti-gst antibody or with an antibody specific for the target protein. Preblocked plates prevent nonspecific binding for highly sensitive results with low background. Versatile format provides 12 strips of eight wells for processing 8-96 samples at one time. Unused strips can be safely stored until needed. Reagents are also available for performing Western blot analysis using anti-gst antibody. This technique uses only 5-10 µl of sample and is highly specific. The method also provides information on the size of the protein. For detailed methodologies, see our technical handbooks listed on page

12 Fusions without the confusion Choose the GST Gene Fusion System to make your research more effective Take away the worry and effort from the production of fusion proteins and focus on the important parts of your research. Optimized and standardized, around the world The components of GST Gene Fusion System work together, using a standardized methodology. This means less work in devising experimental protocols. And if you are part of a consortium, you can obtain and use the same vector constructs methodology and equipment, and rely on getting comparable results, anywhere around the world. All from one Amersham Biosciences supplies all the reagents and instruments you need for producing, detecting, and purifying GST fusion proteins. And when you want to scale up your production, we offer purification media and columns to suit a wide range of vectors, equipment, and the most sensitive detection systems. We also offer handbooks to help you in your research. The right choice GST Gene Fusion System is the right choice for producing and purifying fusion proteins. A wide range of vectors, various purification systems, and a sensitive detection system all mean that you can obtain large yields of high-quality target proteins with minimum difficulty. 12

13 Find out more about the methodology Recombinant Protein Handbook (Code No ) Covers topics including GST and His fusion proteins, protein amplification, extraction, purification, handling of inclusion bodies, methodology, applications, and technical information. GST Gene Fusion Handbook (Code No ) Covers topics including cloning, monitoring, purification, detection, and applications of GST fusion proteins. 13

14 Ordering information Product Quantity Code No. Cloning and expression pgex-6p-1 25 µg pgex-6p-2 25 µg pgex-6p-3 25 µg pgex-5x-1 25 µg pgex-5x-2 25 µg pgex-5x-3 25 µg pgex-4t-1 25 µg pgex-4t-2 25 µg pgex-4t-3 25 µg pgex-2tk 25 µg E. colibl21 (All pgex vectors include E. colib21) 1 vial Purification Prepacked columns and media GST MicroSpin Purification Module 50 purifications Glutathione Sepharose 4B 10 ml ml ml GSTrap FF 2 1 ml ml ml ml GSTPrep FF 16/ ml Glutathione Sepharose 4 Fast Flow 25 ml ml ml Removal of trypsin-like serine proteases HiTrap Benzamidine FF (high sub) 2 1 ml ml ml Benzamidine Sepharose 4 Fast Flow (high sub) 25 ml Detection GST 96-Well Detection Module 96 reactions GST Detection Module 50 reactions Anti-GST Antibody 50 detections Anti-GST HRP Conjugate 75 µlrnp1236 ECL GST Western Blotting Detection Kit 1 RPN1237 Cleavage enzymes PreScission Protease 500 units Factor Xa 400 units Thrombin 500 units Related products ECL Western Blotting ECL Advance Western Blotting Detection Kit For 1000 cm 2 membrane RPN2135 ECL Plus Western Blotting Detection Reagents For 1000 cm 2 membrane RPN2132 ECL Plus Western Blotting Reagent Pack 1 RPN2124 ECL Blocking Agent 1 RPN2125 Hybond Blotting Membrans Hybond -P 10 sheets RPN2020F Hybond ECL 10 sheets RPN2020D 14

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16 For more information about our complete proteomics offering, visit: ÄKTAprime, ECL, ECL Advance, ECL Plus, GSTPrep, GSTrap, HiPrep, HiTrap, Hybond, MicroSpin, PreScission, and Sepharose are trademarks of the Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc. Licensing Information All materials for research use only. Not for diagnostic or therapeutic purposes. A license for commercial use of GST gene fusion vectors must be obtained from Chemicon International Incorporated, Single Oak Drive, Temecula, California 92590, USA. Amersham Biosciences AB Björkgatan 30, SE Uppsala, Sweden Amersham Biosciences UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Biosciences Corp 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855, USA Amersham Biosciences Europe GmbH Munzinger Strasse 9, DE Freiburg, Germany Amersham Biosciences K.K. Sanken Building, , Hyakunincho, Shinjuku-ku, Tokyo , Japan All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them. A copy of these terms and conditions is available on request. Amersham Biosciences AB All rights reserved