A new approach for alteration of protease functions: pro-sequence engineering

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Proteases, like subtilisins, require N-terminal pro-sequences for proper folding and activation.
• Pro-sequences are typically degraded after folding, as they are not essential for mature enzyme function.
• The pro-sequence acts as an intramolecular chaperone, crucial for guiding the correct folding of the protease domain.

Purpose of the Study:

• To investigate the role of the pro-sequence in protease folding and function.
• To explore the potential of pro-sequence engineering for modifying protease properties.
• To introduce the concept of 'pro-sequence engineering' as a novel protein engineering strategy.

Main Methods:

• Utilizing site-directed and random mutagenesis to alter subtilisin pro-sequences.
• Creating chimeric proteases and employing gene shuffling within the subtilisin family.
• Investigating the chaperone activity of pro-sequences from homologous subtilisins.

Main Results:

• Pro-sequence modifications improved autoprocessing efficiency in mutant subtilisins with altered substrate specificities.
• A pro-sequence from a subtilisin homologue successfully chaperoned the refolding of denatured subtilisin.
• Demonstrated that engineering the pro-sequence can enhance protease function and alter substrate specificity.

Conclusions:

• Pro-sequence engineering is a viable strategy for improving autoprocessing proteases.
• This approach offers a new avenue for protein engineering, distinct from traditional methods focused on the protease domain.
• Pro-sequence engineering serves as a valuable tool for understanding protein folding mechanisms and developing novel proteases.