What is the size in kDa of the GST MLF1 fusion protein?
A 46-kDa full-length 35S-labeled MLF1IP protein was produced by in vitro transcription/translation, IVT- MLF1IP, and used to investigate its interaction with GST-MLF1.
What small molecule does glutathione S transferase GST have a high affinity for?
GSH
GST-tags and the GST pull-down assay Because the GST protein has a strong binding affinity for GSH, beads coated with the compound can be added to the protein mixture; as a result, the protein of interest attached to the GST will stick to the beads, isolating the protein from the rest of those in solution.
How big is the GST tag?
26 kDa
At 26 kDa, GST is considerably larger than many other fusion protein affinity tags.
What does glutathione S transferase do?
Glutathione S-transferases (GSTs) are ubiquitous enzymes that are encoded by a large gene family, and they contribute to the detoxification of endogenous or xenobiotic compounds and oxidative stress metabolism in plants.
Why is GST used as a fusion protein?
The use of GST as a fusion tag is desirable because it can act as a chaperone to facilitate protein folding, and frequently the fusion protein can be expressed as a soluble protein rather than in inclusion bodies.
What is GST fusion protein?
In nature, the GST (Glutathione-S-transferase) protein is an enzyme that catalyzes the protective mechanisms of glutathione (GSH). Glutathione is an antioxidant that prevents cell damage by reactive oxygen species. A gst fusion protein is a protein that is tagged with GST protein.
How do you elute GST fusion protein?
Elute the fusion protein by resuspending the resin with 1.0 ml glutathione elution buffer per ml bed volume. Incubate 10 min at room temperature. Centrifuge 500 × g for 5 min. Transfer the fusion protein-containing supernatant to a separate tube.
What is GST protein purification?
What is GST-tagged protein purification? Recombinant proteins that have Glutathione S-transferase (GST) tags are purified using affinity chromatography. GST binds strongly and specifically to chromatography resins coupled with glutathione.
How does GST bind to glutathione?
The 26KDa GST moiety binds with high affinity to glutathione coupled to a Sepharose matrix. This binding is reversible and the protein can be eluted under mild, non-denaturing conditions by the addition of reduced glutathione to the elution buffer.
How do I purify GST tagged protein?
GST protein from various sources, both native and recombinantly expressed in Escherichia coli and other host cells, can be purified by affinity chromatography on immobilized glutathione, followed by competitive elution with excess reduced glutathione (Smith et al., 1988).
Why is DTT added to PBS?
DTT is a reducing agent and usage will ensure that the protein is unfolded and soluble, easy to purify. To keep the cysteine side chains in their normal reduced state, a reducing agent such as DTT is included in the purification.
Why is GST used?
The main objective of incorporating the GST was to eliminate tax on tax, or double taxation, which cascades from the manufacturing level to the consumption level. For example, a manufacturer that makes notebooks obtains the raw materials for, say, Rs. 10, which includes a 10% tax. This means that they pay Rs.
What is the use of GST tag in protein–protein interactions?
This technique can be used to elucidate direct protein–protein interactions. A drawback of this assay is that the protein of interest is attached to GST, altering its native state. A GST-tag is often used to separate and purify proteins that contain the GST-fusion protein.
What are the drawbacks of a GST-tag assay?
A drawback of this assay is that the protein of interest is attached to GST, altering its native state. A GST-tag is often used to separate and purify proteins that contain the GST-fusion protein. The tag is 220 amino acids (roughly 26 kDa) in size, which, compared to tags such as the Myc-tag or the FLAG-tag, is quite large.
What is a GST tag?
The GST tag. GST is a 211 amino acid protein (26 kDa) whose DNA sequence is frequently integrated into expression vectors for production of recombinant proteins. The result of expression from this vector is a GST-tagged fusion protein in which the functional GST protein (26 kDa) is fused to the N-terminus of the recombinant protein.
How are GST-tagged fusion proteins purified or detected?
In addition, GST-tagged fusion proteins can be purified or detected based on the ability of GST (an enzyme) to bind its substrate, glutathione (GSH). Glutathione is a tripeptide (Glu-Cys-Gly) that is the specific substrate for glutathione S-transferase (GST).