Cleavable Self-Aggregating Tags (cSAT) for Therapeutic Peptide Expression and Purification

Area of Science:

• Biotechnology
• Protein and Peptide Engineering
• Biochemistry

Background:

• Therapeutic peptides are crucial but challenging to produce biologically.
• Existing methods struggle with unstable or difficult-to-express peptides.
• Need for efficient and cost-effective peptide production technologies.

Purpose of the Study:

• To develop a facile, reliable, and cost-effective peptide production and purification strategy.
• To enable the expression and purification of therapeutic peptides with authentic N-termini.
• To address challenges in producing peptides prone to degradation.

Main Methods:

• Utilized short self-assembling peptides (e.g., L6KD) and a C-terminal cleavage intein (e.g., Mtu ΔI-CM).
• Developed a cleavable self-aggregating tag (cSAT) scheme for in vivo aggregate formation.
• Employed centrifugation for aggregate separation and pH-induced intein self-cleavage for peptide release.

Main Results:

• Achieved a yield of approximately 4.4 μg/mg wet cell pellet for the therapeutic peptide GLP-1.
• Demonstrated successful purification of peptides with authentic N-termini.
• Validated the cSAT strategy for peptides in the 30-100 amino acid range.

Conclusions:

• The cSAT strategy offers a viable approach for producing peptides, especially those difficult to express.
• This method is cost-effective and yields peptides with native N-termini.
• Facilitates the biotechnological production of therapeutic peptides previously limited by expression challenges.