pGEX Plasmids
pGEX plasmids are a family of expression vectors designed for high-yield production of glutathione-S-transferase (GST)-fusion proteins in Escherichia coli. The fusion of GST to a target protein provides a convenient way to enhance solubility, simplify purification through glutathione affinity chromatography, and enable functional assays. These plasmids are widely used in molecular biology, biochemistry, and structural biology for studying protein function, interactions, and structure.
Key Features of pGEX Plasmids
- Multiple Cloning Site (MCS):
- Located downstream of the GST sequence, enabling the insertion of the gene of interest to create a GST-fusion protein.
- Thrombin or Factor Xa Cleavage Sites:
- Included between the GST tag and the MCS to allow removal of the GST tag after purification if desired.
- Ampicillin Resistance (ampR):
- Provides selection in E. coli using ampicillin.
- Replication Origin (Ori):
- Derived from pBR322, allowing moderate plasmid copy number.
- Size:
- Approximately 4.9–5.0 kb, depending on the specific variant.
Applications of pGEX Plasmids
- Protein Purification:
- Simplifies purification of recombinant proteins using glutathione-Sepharose affinity chromatography.
- Protein Solubility Enhancement:
- GST tag improves the solubility of difficult-to-express proteins, reducing aggregation in E. coli.
- Protein-Protein Interaction Studies:
- GST-fusion proteins are used in pull-down assays to identify binding partners and study interactions.
- Enzymatic Activity Studies:
- Retains enzymatic activity of GST, enabling fusion proteins to bind and release from glutathione-based resins under mild conditions.
- Antibody Production:
- GST-fusion proteins are used as antigens for generating specific antibodies.
pGEX plasmids are highly effective tools for expressing, purifying, and studying recombinant proteins in E. coli. Their GST-fusion system simplifies purification, enhances solubility, and supports versatile downstream applications, such as functional and structural analyses. These plasmids are a mainstay in molecular biology and biochemistry research, offering reliable and scalable protein expression solutions.
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