Extraction and separation of proteins by ionic liquid aqueous two-phase system

Area of Science:

• Biochemistry
• Analytical Chemistry
• Separation Science

Background:

• Protein purification is crucial for bioactive substance isolation.
• Traditional methods can be inefficient or denature proteins.
• Ionic liquid aqueous two-phase systems (IL-ATPS) offer a novel approach for protein separation.

Purpose of the Study:

• To establish a robust protocol for protein extraction and separation using IL-ATPS.
• To identify the optimal ionic liquid and conditions for efficient protein partitioning.
• To evaluate the method's effectiveness for purifying various proteins while maintaining activity.

Main Methods:

• Screening of eight ionic liquids, selecting 1-octyl-3-methylimidazolium bromide ([omim][Br]).
• Single-factor experiments and investigative tests to determine optimal extraction conditions.
• Utilizing proteins with different isoelectric points (BSA, Hb, Lys) and assessing enzyme activity (Try).
• Analysis of linearity, LOD, RSD, repeatability, and precision for quantification.

Main Results:

• Optimal conditions were identified for protein extraction using [omim][Br].
• High average recoveries (90.5%-94.5%) and low RSDs (<1.34%) were achieved for BSA, Hb, Try, and Lys.
• The method demonstrated excellent linearity (R² > 0.9985), low LODs, and good stability and precision.
• Protein activity was confirmed, and a protein-IL clustering phenomenon was observed and evaluated.

Conclusions:

• A satisfactory IL-ATPS protocol was established for protein purification.
• The method shows potential for broad application in purifying diverse bioactive substances.
• The observed clustering phenomenon is a key factor in protein partitioning and offers valuable insights.