Catalytically Active Bioseparation Resin Utilizing a Covalent Intermediate for Tagless Protein Purification

Area of Science:

• Biotechnology
• Protein Biochemistry
• Molecular Biology

Background:

• Traditional protein purification methods using noncovalent affinity binding have limitations including target leakage and the need for tag removal.
• Existing methods often restrict wash stringency, impacting impurity removal and overall purification efficiency.

Purpose of the Study:

• To develop an innovative protein purification method overcoming the limitations of current noncovalent affinity-based techniques.
• To engineer a novel resin system for efficient and tagless protein isolation.

Main Methods:

• Developed the Catch, Hold, and Release (C-H-R) method utilizing a single-use resin composed of polyhydroxybutyrate beads.
• The resin co-displays SpyCatcher (SpyC) domains and Sortase A (SrtA), assembled within engineered Escherichia coli.
• SpyTagged (SpyT) proteins with an SrtA cleavage site were ligated to SpyC, and inducible SrtA catalyzed tagless protein release.

Main Results:

• Successfully purified three diverse proteins from complex mixtures with high purity and yields.
• The C-H-R method establishes a covalent link to the target protein, preventing leakage during purification.
• Inducible Sortase A enabled efficient release of pure, tagless target proteins while the SpyT remained bound.

Conclusions:

• The Catch, Hold, and Release (C-H-R) method represents a novel protein purification concept utilizing a covalent linkage.
• This innovative bioseparation technique offers improved purity, yield, and efficiency compared to traditional methods.
• The C-H-R system provides a robust platform for tagless protein purification from complex biological samples.