How Is GST Fusion Protein Purified?

GST fusion proteins, which consist of a target protein fused to glutathione S-transferase (GST) via genetic engineering, are commonly used to facilitate protein expression and purification. The purification process relies on the high-affinity interaction between GST and glutathione (GSH), and is typically performed using glutathione-based affinity chromatography.

 

The standard protocol for GST fusion protein purification involves the following steps:

 

Transformation of Expression Vector

Introduce the expression vector encoding the GST-tagged target protein into a suitable host, typically Escherichia coli.

 

Induction of Protein Expression

Use an inducer such as IPTG to trigger the expression of the GST fusion protein in the host cells.

 

Cell Lysis

Harvest the induced cells and lyse them by sonication, high-pressure homogenization, or enzymatic treatment to release the intracellular contents.

 

Clarification of Lysate

Remove cellular debris and insoluble material via centrifugation or filtration to obtain a clear lysate.

 

Glutathione Affinity Chromatography

Apply the clarified lysate to a pre-equilibrated glutathione Sepharose column. GST-tagged proteins will bind specifically to the immobilized glutathione, while non-specifically bound proteins are removed through washing steps.

 

Elution of Gst Fusion Proteins

Elute the bound fusion proteins using a buffer containing a high concentration of reduced glutathione, which competitively displaces the GST from the column matrix.

 

Target Protein Recovery (Optional)

If the aim is to isolate the target protein alone, cleave the fusion protein using site-specific proteases (e.g., TEV protease or thrombin) to release the target from the GST tag. Residual GST and protease can then be removed through subsequent chromatographic steps.

 

Purity Assessment and Protein Concentration

Evaluate the purity of the isolated proteins by SDS-PAGE and/or Western blot. If necessary, concentrate the protein using ultrafiltration, gel filtration, or other suitable methods.

 

Protein Storage

Store the purified GST fusion protein or cleaved target protein in an appropriate storage buffer at –20°C or –80°C to maintain stability.

 

GST fusion protein purification via glutathione affinity chromatography offers high specificity and ease of implementation, often yielding high-purity products. However, careful optimization of each step is essential to ensure reproducibility and overall purification efficiency.

 

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

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