A cleavable self-assembling tag strategy for preparing proteins and peptides with an authentic N-terminus

Area of Science:

• Biotechnology
• Protein Chemistry
• Molecular Biology

Background:

• Recombinant protein and peptide production is crucial for biotechnology.
• Existing purification methods can be complex and costly.
• Producing proteins with authentic N-termini is often challenging.

Purpose of the Study:

• To develop a streamlined and economical strategy for protein and peptide purification.
• To create a method yielding target molecules with an authentic N-terminus.
• To improve the purification of unstable peptides.

Main Methods:

• Utilized a fusion protein strategy involving self-assembling peptides and C-terminal cleavage inteins.
• Expressed fusion proteins as aggregates induced by self-assembling peptides.
• Employed intein-mediated cleavage for release of target proteins/peptides.

Main Results:

• Tested combinations of four self-assembling peptides (ELK16, L6 KD, FK, FR) and three inteins (Sce VMA, Mtu ΔI-CM, Ssp DnaB).
• Identified Mtu ΔI-CM intein with L6 KD, FR, and FK peptides as an effective combination.
• Achieved yields comparable to established strategies (e.g., Trx-strategy) with enhanced simplicity and cost-effectiveness.

Conclusions:

• The novel strategy offers a simple and efficient method for purifying proteins and peptides.
• This approach is particularly effective for producing unstable peptides (30-100 amino acids) with authentic N-termini.
• The method simplifies recombinant protein expression and purification processes.