GST-tagged Proteins
The Glutathione S-transferase (GST) fusion system is a comprehensive system to express, purify, and detect GST-tagged proteins. The GST affinity tag allows protein purification under mild conditions and easy cleavage, keeping the proteins in their native structure and function.Three major parts comprise this system: pGEX plasmid expression vectors, GST-tagged protein purification, and GST-tagged protein detection.
Advantage of GST fusion system:
1. All pGEX vectors provide a tac promoter to express proteins at high-level and with chemical induction;
2. Purify active proteins under mild and nondenaturing conditions;
3. Suitable for one-step, high-yield affinity chromatography purification based on glutathione sepharose media;
4. Easy cleavage of target protein from fusion protein with multiple proteases such as Thrombin or Factor Xa;
5. Convenient detection of GST fusion protein with antibodies.
GST-Tag
GST is an antioxidant enzyme composed of 211 amino acids (MW, around 26 kDa). It is water soluble and involves of the primary cellular defense mechanisms a dimer in all aerobic organisms. DNA sequence of GST is frequently applied in plasmid construction. The resulting expression vector is used to produce GST-tagged fusion protein, and GST tag enhance the expression level and solubility of recombinant proteins.
GST-tagged Protein Detection
1. GST 96-Well Detection Module;
This module allows to determine GST fusion proteins in various sample rapidly and sensitively.
2. Anti-GST Antibody;
This polyclonal antibody isolated from the sera of goats can be used to detect recombinant GST-tagged proteins with high sensitivity and specificity.
3. GST Detection Module;
This module permits sensitive determination of GST-tagged proteins based on a biochemical assay or an immunoassay in which glutathione and CDNB used as substrates for GST.
GST-tagged Protein Purification Troubleshooting
| Problem | Cause | Solution |
| Low protein yield | Problems with vector construction | Ensure that protein and tag are in frame |
| Low protein expression | Optimize expression conditions | |
| Inclusion bodies | Lower the growth temperature | |
| Insufficient extraction | Check lysozyme and sonication conditions | |
| Poor binding efficiency | Fusion protein is not well concentrated | Concentrate the sample |
| Flow rate is too high | Decrease flow rate | |
| Reducing agent missing | Add DTT (5 mM) to the lysis buffer | |
| Severe sonication | Use milder sonication conditions | |
| Inefficient elution | Flow rate is too high | Decrease flow rate |
| Low elution volume | Increase the volume of Elution Buffer | |
| Elution Buffer stored for too long | Prepare Elution Buffer immediately before use | |
| Poor protein purity | Insufficient washing | Repeat washing steps |
| Fusion protein degradation | Add a protease inhibitor or perform purification on ice | |
| Severe sonication | Use milder sonication conditions |
Creative Biostructure is pleased to accelerate your GST-tagged protein research project from expression vector construction to high-yield recombinant protein production. Please see the table below for more information and feel free to Contact Us.
