Biotinylated-sortase self-cleavage purification (BISOP) method for cell-free produced proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Affinity tags are crucial for recombinant protein purification but can hinder structural and functional analyses.
• Existing tag removal methods often involve protease treatment, which can be inefficient.
• Previous self-cleavage methods were not adapted for cell-free protein production systems.

Purpose of the Study:

• To develop an efficient method for producing tag-free recombinant proteins using a cell-free system.
• To overcome limitations of existing purification techniques in cell-free protein synthesis.
• To enable high-quality protein production for biochemical and structural analyses.

Main Methods:

• Initial attempts utilized a histidine tag-based sortase self-cleavage system in a wheat cell-free synthesis, which faced challenges with cleavage and recovery.
• The system was optimized by using biotin as an affinity tag, leading to the development of the biotinylated sortase self-cleavage purification (BISOP) method.
• The BISOP method was validated using Green Fluorescent Protein (GFP) and successfully purified protein complexes and active enzymes.

Main Results:

• The histidine tag-based method showed high background and low recovery, with unintended cleavage.
• The BISOP method significantly improved protein recovery and yielded highly purified, tag-free proteins.
• N-terminal sequencing confirmed correct cleavage, and the method was used to purify protein complexes and active kinases.
• The BISOP method demonstrated suitability for automated protein synthesis and purification.

Conclusions:

• The newly developed BISOP method is highly effective for obtaining high-quality, tag-free recombinant proteins from cell-free systems.
• This method is valuable for subsequent biochemical and structural analyses.
• BISOP offers a robust solution for tag-free protein purification in automated workflows.