An efficient thermostabilization strategy based on self-assembling amphipathic peptides for fusion tags

Area of Science:

• Biochemistry
• Protein Engineering
• Biotechnology

Background:

• Self-assembling amphipathic peptides (SAPs) are utilized as tags to enhance enzyme stability, but their efficacy varies across different enzymes.
• Understanding the molecular determinants of SAP-mediated stabilization is crucial for optimizing their application.

Purpose of the Study:

• To identify key factors governing SAP-mediated enzyme stabilization.
• To develop a comprehensive strategy for enhancing protein stability using a library of engineered SAPs and optimized conditions.
• To demonstrate the efficacy of this strategy on various enzymes.

Main Methods:

• Investigated the impact of SAP length and linker properties on enzyme stabilization using S1 (AEAEAKAK)2 and polygalacturonate lyase (PGL).
• Analyzed the mechanism of SAP stabilization, revealing induction of loose protein oligomerization via intermolecular hydrophobic interactions.
• Developed a library of stabilizing tags by combining different SAPs and linker peptides, and optimized conditions using sodium chloride (NaCl).

Main Results:

• Identified SAP length and linker length/flexibility as critical for stabilization.
• Demonstrated that SAPs promote enzyme stabilization through intermolecular hydrophobic interactions leading to oligomerization.
• Achieved significant increases in half-life (t1/2) for PGL (33.25-fold), lipoxygenase (LOX, 17.55-fold), and L-asparaginase (15.6-fold) using the developed strategy.

Conclusions:

• A comprehensive strategy involving engineered SAP libraries and NaCl treatment effectively enhances enzyme stability.
• The findings provide a robust platform for designing stabilized enzymes with improved half-lives for various applications.
• The developed SAP library exhibits significant potential for broad application in enzyme and protein stability enhancement.