Tuning permeabilization of microbial cells by three-phase partitioning

Area of Science:

• Biotechnology
• Protein Chemistry
• Cell Biology

Background:

• Cell permeabilization is crucial for intracellular product recovery.
• Traditional methods often lack selectivity and efficiency.
• Developing selective cell disruption techniques is essential for bioprocessing.

Purpose of the Study:

• To investigate the efficacy of three-phase partitioning (TPP) for selective protein release.
• To optimize TPP by incorporating a preincubation step for enhanced selectivity.
• To demonstrate the applicability of TPP across different microbial species.

Main Methods:

• Three-phase partitioning using t-butanol and ammonium sulfate.
• Preincubation of microbial cells (Thermus thermophilus, Saccharomyces cerevisiae, Escherichia coli) with t-butanol.
• Analysis of protein release based on molecular weight and preincubation time.
• Verification of protein purity using specific activity and electrophoresis.

Main Results:

• TPP successfully permeabilized cells, forming an interface between aqueous and alcohol layers.
• Preincubation time modulated protein release selectivity.
• Proteins < 34 kDa were released within 15 min, while ~85 kDa proteins required 30 min.
• High-molecular-weight proteins (>150 kDa) were retained even after 60 min.
• Obtained proteins exhibited high purity, confirmed by specific activity and electrophoretic analysis.

Conclusions:

• Three-phase partitioning with a t-butanol preincubation step offers a tunable and selective method for protein extraction.
• This technique allows for the separation of proteins based on molecular weight from various microbial sources.
• TPP is a promising tool for efficient and high-purity protein recovery in biotechnology.