A general purification platform for toxic proteins based on intein trans-splicing

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Overexpression of functional proteins can lead to host cell toxicity in recombinant systems.
• This toxicity hinders the characterization and production of valuable proteins.
• Existing methods for toxic protein expression often fail with highly toxic targets.

Purpose of the Study:

• To develop a novel protein purification platform for producing proteins toxic to recombinant hosts.
• To leverage intein trans-splicing for controlled expression and purification of challenging proteins.
• To engineer a hybrid split intein for enhanced compatibility and efficiency.

Main Methods:

• A split intein system was employed, dividing the target toxic protein into two inactive fragments.
• Fragments were expressed fused to complementary intein domains, immobilized on an affinity column.
• Intein-mediated trans-splicing was induced to release the mature, purified toxic protein.
• A hybrid split intein (Npu DnaE N-terminus, Ssp DnaE C-terminus) was engineered for improved splicing tolerance.

Main Results:

• The developed platform successfully purified a highly toxic homing endonuclease (I-TevI).
• The engineered hybrid split intein demonstrated increased tolerance at the splicing junction.
• Protein yields using the hybrid intein were 50% higher compared to the native Npu DnaE intein.
• Purified I-TevI protein exhibited native biological activity.

Conclusions:

• Intein trans-splicing offers a robust strategy for producing extremely toxic proteins.
• The engineered hybrid split intein enhances the efficiency and applicability of this purification method.
• This platform significantly advances recombinant protein production for challenging toxic targets.