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

Abstract

Efficient protein and peptide expression and purification technologies are highly needed in biotechnology, especially in light of the increasing number of proteins and peptides that are being exploited for therapeutic use, which are inherently difficult to produce via biological means. In this chapter, we describe a facile, reliable, and cost-effective peptide production and purification strategy based on short self-assembling peptides (e.g., L₆KD (LLLLLLKD)) and a C-terminal cleavage intein (e.g., Mtu ΔI-CM). This cleavable self-aggregating tag (cSAT) scheme depends on the in vivo formation of aggregates of the fusion protein containing the target peptide, which is induced during the expression by the presence of the self-assembling peptide in the construct. After a simple separation of the aggregates by centrifugation, the purified target peptide with authentic N-terminus is released in solution by pH-induced intein self-cleavage. As an example, a yield of about 4.4 μg/mg wet cell pellet was obtained when the cSAT scheme was used for the expression and purification of the therapeutic peptide GLP-1. This strategy provides a viable approach for preparing peptides with authentic N-termini, especially those in the range of 30 ~ 100 amino acids in size that are typically unstable or susceptible to degradation in Escherichia coli.