Cleaving the way for heterologous peptide production: An overview of cleavage strategies

One of the main bottlenecks for recombinant peptide production is choosing the proper cleavage method to remove fusion protein tags from target peptides. While these tags are crucial for inhibiting the activity of the target peptide during heterologous expression, incorporating a cleavage site is essential for their later removal, ensuring the pure sequencing of the peptide. This review evaluates different cleavage methods, including protease-mediated, self-cleavable protein, and chemical-mediated sites, regarding their advantages and limitations. For instance, intein, N^pro EDDIE, enterokinase, factor Xa, SUMO, and CNBr are options for residue-free cleavage. Although protease-mediated cleavage is widely used, it can be expensive, due to its own cost added to the whole process. As an alternative, self-cleavable sites eliminate the requirement for proteinases. Another crucial step in defining the proper cleavage method is cost consideration, which relates to the purpose of peptide production. Here, we explore a range of cleavage approaches, meeting the needs of both cost-constrained applications and a more flexible budget. Overall, selecting the most suitable cleavage method should be based on careful consideration of toxicity, cost, accuracy, and specific application requirements to ensure a state-of-the-art approach.