Improved self-cleaving precipitation tags for efficient column free bioseparations.

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Current protein purification methods often involve multiple steps, expensive reagents, and can result in low yields.
• Conventional affinity tag systems require specific resins and proteolytic enzymes for tag removal, adding complexity and cost.
• Existing self-cleaving tag systems can suffer from premature cleavage, leading to product loss during expression.

Purpose of the Study:

• To evaluate novel Mtu RecA ΔI-CM mini-intein mutants for enhanced protein purification.
• To assess the performance of these mutants with elastin-like polypeptide (ELP) precipitation tags.
• To develop a more efficient and cost-effective single-step protein purification strategy.

Main Methods:

• Yeast surface display was used to generate and select novel mini-intein mutants.
• The performance of the selected mutants (ΔI-12 and ΔI-29) was evaluated using three model proteins: GFP, MBP, and beta-gal.
• Purification efficiency, precursor content, product purity, and cleavage efficiency were quantified.

Main Results:

• The novel mini-intein mutants (ΔI-12 and ΔI-29) significantly improved precursor content, product purity, and process yield compared to the original mini-intein.
• Single-step purification achieved product purities ranging from 68% to 94% for the model proteins.
• High cleavage efficiency was observed within 5 hours under various conditions.

Conclusions:

• The developed mini-intein mutants represent improved self-cleaving precipitation tags for protein purification.
• These tags enable efficient, single-step purification of diverse proteins with high yields and purity.
• This technology offers a cost-effective solution for laboratory-scale protein purification.