Rapid, continuous purification of proteins in a microfluidic device using genetically-engineered partition tags

Area of Science:

• Biotechnology
• Protein Purification
• Microfluidics

Background:

• High-throughput screening assays require minute amounts of purified proteins.
• Existing protein purification methods can be time-consuming and require large sample volumes.

Purpose of the Study:

• To develop a rapid, automated microscale process for isolating specific proteins from E. coli cell lysate.
• To improve protein recovery and purity for applications in drug screening and enzyme engineering.

Main Methods:

• Developed a microfluidic device utilizing a flowing aqueous two-phase system.
• Genetically tagged recombinant proteins to drive partitioning into the PEG-rich phase.
• Integrated cell culture, lysis, purification, and analysis on a single lab-on-a-chip device.

Main Results:

• The process removed approximately 85% of contaminating proteins, nucleic acids, and cell debris.
• Genetic tagging tripled recovery of AcGFP1 and increased glutathione S-transferase (GST) recovery to 40%.
• The mild two-phase system preserved protein function, with active enzymes and functional fluorescent proteins recovered.

Conclusions:

• This microfluidic aqueous two-phase extraction offers a rapid, single-step purification method.
• The developed lab-on-a-chip device streamlines protein purification without expensive affinity reagents or chromatography.
• This automated microscale purification is suitable for high-throughput screening and protein engineering applications.