Proteomics

What is the advantage of using recombinant DNA technology in protein production?

• precise manipulation of DNA sequences

• expression of proteins in various host organisms

• perfect recombinant DNA every time

• Decreased antibiotic resistance

What is the purpose of expressing a protein as a fusion construct containing an epitope tag?

• to express the protein at high levels

• to denature the protein

• to purify the protein

• to create an inter-membrane protein

What type of affinity tags are commonly used in IMAC?

• GFP

• Glutathione S-transferase-tag

• poly-adenine tails

• His-tag

What does IMAC stand for?

Answer explanation

Immobilised Metal Affinity Chromatography

What is the basic principle behind IMAC?

Answer explanation

To selectively isolate a target protein from a complex mixture based on its specific interaction with an immobilized ligand. Affinity chromatography exploits the high affinity and specificity of interactions between the target protein and its ligand, allowing for highly efficient purification with minimal non-specific binding.

what is the significance/function of Nickel in the Ni-NTA resin?

Answer explanation

Nickel binds to the His tag with high affinity via coordination bonds.

what is the difference between native and non-native protein purification?

Answer explanation

• Native: Aims to isolate the protein in its natural form, preserving its structural integrity and biological activity. Often employed for studying protein function, interactions with other molecules, and potential use in therapeutic applications. Milder purification techniques are crucial to minimize alterations to the protein's structure and post-translational modifications (PTMs) that might be crucial for its function.

Non-native: Focuses on isolating the protein in high yield and purity, often prioritizing quantity over maintaining the protein's native state.

• Commonly used for protein characterization, structural studies, and large-scale production.

• Harsher techniques might be employed, potentially affecting the protein's conformation and PTMs.

Native protein purification can be exploited to -- proteins such as enzyme subunits and binding proteins.

What is needed for successful his-tag protein purification under native conditions

• Presence of inclusion bodies

• Solubility of the 6xHis-tagged protein

• Denaturation of the protein

• Binding to the Ni-NTA resin only

Which factor contributes to nonspecific binding of proteins in native conditions during affinity purification?

• Presence of inclusion bodies

• pH near the isoelectric point

• Denaturation of the protein

• Low concentration of imidazole in buffers