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This study enhances lysozyme crystallization yield using liquid-liquid phase separation (LLPS) with NaCl and HEPES additives. This method achieves over 90% yield in one hour, offering a sustainable protein purification alternative.

Keywords:
HEPESLLPSNaCl

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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.