The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins

Area of Science:

• Protein engineering
• Biochemistry
• Synthetic biology

Background:

• Inteins are protein segments that catalyze their own excision from precursor proteins.
• Modified inteins can be engineered for specific ligation and cyclization strategies.
• Protein cyclization is a valuable technique for enhancing peptide stability and function.

Purpose of the Study:

• To develop a versatile two-intein system for protein ligation and cyclization.
• To demonstrate the system's capability in producing cyclic peptides and protein polymers.
• To explore the potential of this system for biomaterial development.

Main Methods:

• Cloning of modified Synechocystis sp. (Ssp DnaB intein) and Mycobacterium xenopi (Mxe GyrA intein) in-frame with target proteins.
• Induction of peptide bond cleavage to generate N-terminal cysteine and C-terminal thioester.
• Intra- or intermolecular condensation for cyclization or polymerization.
• Mass spectrometry for verifying the molecular weights of cyclic peptides.

Main Results:

• Successful generation of cyclic peptides (BBP, RGD, CDR-H3/C2) with accurate mass verification.
• Demonstrated cyclization of proteins up to 395 amino acids.
• Formation of thioredoxin multimers, indicating potential for fibrous protein biomaterials.

Conclusions:

• The developed two-intein system is effective for creating cyclic peptides and large cyclic proteins.
• This technology holds promise for the development of novel protein-based biomaterials.
• The system offers a powerful tool for protein engineering and synthetic biology applications.