Yield of Protein Crystallization from Metastable Liquid-Liquid Phase Separation

Area of Science:

• Biochemistry
• Biotechnology
• Crystallography

Background:

• Protein crystallization is a key purification method but often slow and unreproducible.
• Liquid-liquid phase separation (LLPS) offers a promising strategy to enhance protein crystallization.
• Current methods lack efficiency and reproducibility for large-scale protein purification.

Purpose of the Study:

• To enhance lysozyme crystallization yield using a combination of additives under LLPS conditions.
• To investigate the role of NaCl and HEPES in promoting protein crystallization.
• To establish a rapid and reproducible method for protein purification.

Main Methods:

• Inducing LLPS in lysozyme solutions by lowering temperature with NaCl.
• Utilizing HEPES to stabilize the protein-rich phase and lysozyme crystals.
• Measuring crystallization yield as a function of time and temperature relative to LLPS.

Main Results:

• Achieved over 90% lysozyme crystallization yield within one hour using NaCl and HEPES under LLPS conditions.
• The combined additives yielded a three-fold increase in crystallization compared to NaCl alone.
• Crystallization yield significantly increased when crystallization temperature intersected with LLPS temperature.

Conclusions:

• The combination of NaCl and HEPES under LLPS conditions significantly enhances lysozyme crystallization yield.
• LLPS is an effective strategy for boosting protein crystallization efficiency and reproducibility.
• This approach offers a sustainable and economically viable alternative for protein purification in biotechnology.