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Last updated:09/02/2010

pGEX Vectors (GST Gene Fusion System)

  • A tac promoter for chemically inducible, high-level expression of GST-tagged recombinant proteins.
  • An internal lacIq gene for use in any E. coli host.
  • Very mild elution conditions for release of fusion proteins from the affinity matrix, thus minimizing effects on antigenicity and functional activity.
  • PreScission™ Protease, Thrombin, or Factor Xa recognition sites for cleaving the desired protein from the fusion product.
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* E. coli BL21 is not supplied with the vector. E. coli BL21 can be ordered separately, see E. coli BL21.
† E. coli BL21 included with vector.

pGEX Vectors (GST Gene Fusion System)

Technical Information


Click here to view this image in high resolution
Map of the glutathione S-transferase fusion vectors showing the reading frames and main features. Even though stop codons in all three frames are not depicted in this map, all thirteen vectors have stop codons in all three frames downstream from the multiple cloning site.

Thirteen pGEX vectors are available with or without E. coli BL21 (see Figure). Nine of the vectors have an expanded multiple cloning site (MCS) that contains six restriction sites. The expanded MCS facilitates the unidirectional cloning of cDNA inserts obtained from libraries constructed using many available lambda vectors. pGEX-6P-1, pGEX-6P-2, and pGEX-6P-3 each encode the recognition sequence for site-specific cleavage by PreScission™ Protease, (see PreScission Protease) between the GST domain and the multiple cloning site. pGEX-4T-1, pGEX-4T-2, and pGEX-4T-3 are derived from pGEX-2T and contain a Thrombinsee Thrombin recognition site. pGEX-5X-1, pGEX-5X-2, and pGEX-5X-3 are derivatives of pGEX-3X and possess a Factor Xa see Factor Xarecognition site.

Download the pGEX sequence map in PDF format. For ASCII format please scroll down.

pGEX-2TK is uniquely designed to allow the detection of expressed proteins by directly labeling the fusion products in vitro (1). This vector contains the recognition sequence for the catalytic subunit of cAMP-dependent protein kinase obtained from heart muscle. The protein kinase site is located between the GST domain and the MCS. Expressed proteins can be directly labeled using protein kinase and [g-P32]ATP and readily detected using standard radiometric or autoradiographic techniques. pGEX-2TK is a derivative of pGEX-2T; its fusion proteins can be cleaved with Thrombin.

Cleavage of pGEX-6P GST fusion proteins occurs between the Gln and Gly residues of the recognition sequence Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro (2). Low temperature (5°C) digestion minimizes the degradation of the protein of interest. Because PreScission Protease has been engineered with a GST tag, it can also be removed from the cleavage mixture simultaneously with the GST portion of the fusion protein. The pGEX-6P Expression Vectors permit convenient site-specific cleavage and simultaneous purification on Glutathione Sepharose™. The pGEX-6P series provides all three translational reading frames linked between the GST coding region and the multiple cloning site.

Collectively, the pGEX vectors provide all three translational reading frames beginning with the EcoR-I restriction site. pGEX-1lT, pGEX-6P-1, pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from l gt11 libraries.

VectorUnformattedFormattedGenBank Accession No.
pGEX-1lT, 28-9184-41 AB ASCII PDF U13849 
pGEX-2T, 28-9184-42 AB ASCII PDF U13850 
pGEX-2TK, 28-9189-67 AB ASCII PDF U13851 
pGEX-3X, 28-9184-43 AB ASCII PDF U13852 
pGEX-4T-1, 28-9184-44 AB ASCII PDF U13853 
pGEX-4T-2, 28-9184-45 AB ASCII PDF U13854 
pGEX-4T-3, 28-9184-46 AB ASCII PDF U13855 
pGEX-5X-1, 28-9184-47 AB ASCII PDF U13856 
pGEX-5X-2, 28-9184-48 AB ASCII PDF U13857 
pGEX-5X-3, 28-9184-49 AB ASCII PDF U13858 
pGEX-6P-1, 28-9184-50 AB ASCII PDF U78872 
pGEX-6P-2, 28-9184-51 AB ASCII PDF U78873 
pGEX-6P-3, 28-9184-53 AB ASCII PDF U78874 

Click on “ASCII” to download an unformatted sequence for use by a sequence analysis program. Click on “PDF” to download a formatted sequence and restriction site table. If you prefer accessing the sequence in GenBank, refer to the right-hand column for the GenBank accession number:

  • Expression: Proteins are expressed as fusion proteins with the Mr 26 000 glutathione S-transferase (GST). The GST gene contains an ATG and ribosome-binding site, and is under control of the tac promoter. A translation terminator is provided in each reading frame. The resulting fusion protein may be purified using ( 38861.)
  • Enzymatic cleavage with PreScission Protease: pGEX-6P-1, -2, -3 allow for removal of the GST carrier protein from the fusion protein by enzymatic cleavage with PreScission Protease. Because PreScission Protease has been engineered with a GST tag, it can also be removed simultaneously with the GST portion of the fusion protein.
  • Enzymatic cleavage with Thrombin: pGEX-1lT, pGEX-2T, pGEX-2TK, pGEX-4T-1, -2, -3 allow for removal of the GST carrier protein from the fusion protein by enzymatic cleavage with Thrombin.
  • Enzymatic cleavage with Factor Xa: pGEX-3X, pGEX-5X-1, -2, -3 allow for removal of the GST carrier protein from the fusion protein by enzymatic cleavage with Factor Xa.
  • Direct labeling in vitro: pGEX-2TK allows for direct labeling of fusion proteins in vitro with P32 using the catalytic subunit of cAMP-dependent protein kinase.
  • Host(s): E. coli. The plasmid provides lacIq repressor.
  • Selectable marker(s): Plasmid confers resistance to 100 μg/ml ampicillin.
  • Amplification: Recommended.

Properties of pGEX vectors Induction: tac promoter inducible with 1 to 5 mM IPTG.
pGEX-1lT Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Thrombin cleavage: 918-935
*   MCS: 930-944
*   b-lactamase gene region: Promoter: -10: 1308-1313; -35: 1285-1290; Start codon (ATG): 1355; Stop codon (TAA): 2213
*   lacIq gene region: Start codon (GTG): 3296; Stop codon (TGA): 4376
*   Plasmid replication region: Site of replication initiation: 2973; Region necessary for replication: 2280-2976
*   Sequencing primers: 5' pGEX Sequencing Primer binds nucleotides 869-891; 3' pGEX Sequencing Primer binds nucleotides 1019-997

pGEX-2T Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Thrombin cleavage: 918-935
*   MCS: 930-945
*   b-lactamase gene region: Promoter: -10: 1309-1314; -35: 1286-1291; Start codon (ATG): 1356; Stop codon (TAA): 2214
*   lacIq gene region: Start codon (GTG): 3297; Stop codon (TGA): 4377
*   Plasmid replication region: Site of replication initiation: 2974; Region necessary for replication: 2281-2977
*   Sequencing primers: 5' pGEX Sequencing Primer binds nucleotides 869-891; 3' pGEX Sequencing Primer binds nucleotides 1020-998

pGEX-2TK Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Thrombin cleavage: 918-935;
*   Coding for kinase recognition site: 936-950
*   MCS: 951-966
*   b-lactamase gene region: Promoter: -10: 1330-1335; -35: 1307-1312; Start codon (ATG): 1377; Stop codon (TAA): 2235
*   lacIq gene region: Start codon (GTG): 3318; Stop codon (TGA): 4398
*   Plasmid replication region: Site of replication initiation: 2995; Region necessary for replication: 2302-2998
Sequencing primers: 5' pGEX Sequencing Primer binds nucleotides 869-891; 3' pGEX Sequencing Primer binds nucleotides 1041-1019

pGEX-3X Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Factor Xa cleavage: 921-932
*   MCS: 934-949
*   b-lactamase gene region: Promoter: -10: 1313-1318; -35: 1290-1295; Start codon (ATG): 1360; Stop codon (TAA): 2218
*   lacIq gene region: Start codon (GTG): 3301; Stop codon (TGA): 4381
*   Plasmid replication region: Site of replication initiation: 2978; Region necessary for replication: 2285-2981
*   Sequencing primers: 5' pGEX Sequencing Primer binds nucleotides 869-891; 3' pGEX Sequencing Primer binds nucleotides 1024-1002

pGEX-4T-1 Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Thrombin cleavage: 918-935
*   MCS: 930-966
*   b-lactamase gene region: Promoter: -10: 1330-1335; -35: 1307-1312; Start codon (ATG): 1377; Stop codon (TAA): 2235
*   lacIq gene region: Start codon (GTG): 3318; Stop codon (TGA): 4398
*   Plasmid replication region: Site of replication initiation: 2995; Region necessary for replication: 2302-2998
*   Sequencing primers: 5' pGEX Sequencing Primer binds nucleotides 869-891; 3' pGEX Sequencing Primer binds nucleotides 1041-1019

pGEX-4T-2 Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Thrombin cleavage: 918-935
*   MCS: 930-967
*   b-lactamase gene region: Promoter: -10: 1331-1336; -35: 1308-1313; Start codon (ATG): 1378; Stop codon (TAA): 2236
*   lacIq gene region: Start codon (GTG): 3319; Stop codon (TGA): 4399
*   Plasmid replication region: Site of replication initiation: 2996; Region necessary for replication: 2303-2999
*   Sequencing primers: 5' pGEX Sequencing Primer binds nucleotides 869-891; 3' pGEX Sequencing Primer binds nucleotides 1042-1020

pGEX-4T-3 Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Thrombin cleavage: 918-935
*   MCS: 930-965
*   b-lactamase gene region: Promoter: -10: 1329-1334; -35: 1306-1311; Start codon (ATG): 1376; Stop codon (TAA): 2234
*   lacIq gene region: Start codon (GTG): 3317; Stop codon (TGA): 4397
*   Plasmid replication region: Site of replication initiation: 2994; Region necessary for replication: 2301-2997
*   Sequencing primers: 5' pGEX Sequencing Primer binds nucleotides 869-891; 3' pGEX Sequencing Primer binds nucleotides 1040-1018

pGEX-5X-1 Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Factor Xa cleavage: 921-932
*   MCS: 934-969
*   b-lactamase gene region: Promoter: -10: 1333-1338; -35: 1310-1315; Start codon (ATG): 1380; Stop codon (TAA): 2238
*   lacIq gene region: Start codon (GTG): 3321; Stop codon (TGA): 4401
*   Plasmid replication region: Site of replication initiation: 2998; Region necessary for replication: 2305-3001
*   Sequencing primers: 5’ pGEX Sequencing Primer binds nucleotides 869-891; 3’ pGEX Sequencing Primer binds nucleotides 1044-1022

pGEX-5X-2 Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Factor Xa cleavage: 921-932
*   MCS: 934-970
*   b-lactamase gene region: Promoter: -10: 1334-1339; -35: 1311-1316; Start codon (ATG): 1381; Stop codon (TAA): 2239
*   lacIq gene region: Start codon (GTG): 3322; Stop codon (TGA): 4402
*   Plasmid replication region: Site of replication initiation: 2999; Region necessary for replication: 2306-3002
*   Sequencing primers: 5' pGEX Sequencing Primer binds nucleotides 869-891; 3' pGEX Sequencing Primer binds nucleotides 1045-1023

pGEX-5X-3 Control Regions:
*   Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Coding region for Factor Xa cleavage: 921-932
*   MCS: 934-971
*   b-lactamase gene region: Promoter: -10: 1335-1340; -35: 1312-1317; Start codon (ATG): 1382; Stop codon (TAA): 2240
*   lacIq gene region: Start codon (GTG): 3323; Stop codon (TGA): 4403
*   Plasmid replication region: Site of replication initiation: 3000; Region necessary for replication: 2307-3003
*   Sequencing primers: 5' pGEX Sequencing Primer binds nucleotides 869-891; 3' pGEX Sequencing Primer binds nucleotides 1046-1024

Reference

  1. Kaelin, W.G. et al. Cell 70, 351 (1992).



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